CA2020958C - Nucleic acid sequence amplification methods - Google Patents

Nucleic acid sequence amplification methods Download PDF

Info

Publication number
CA2020958C
CA2020958C CA002020958A CA2020958A CA2020958C CA 2020958 C CA2020958 C CA 2020958C CA 002020958 A CA002020958 A CA 002020958A CA 2020958 A CA2020958 A CA 2020958A CA 2020958 C CA2020958 C CA 2020958C
Authority
CA
Canada
Prior art keywords
rna
primer
dna
sequence
target
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
CA002020958A
Other languages
French (fr)
Other versions
CA2020958A1 (en
Inventor
Daniel L. Kacian
Timothy J. Fultz
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Gen Probe Inc
Original Assignee
Gen Probe Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US07/550,837 external-priority patent/US5480784A/en
Application filed by Gen Probe Inc filed Critical Gen Probe Inc
Publication of CA2020958A1 publication Critical patent/CA2020958A1/en
Application granted granted Critical
Publication of CA2020958C publication Critical patent/CA2020958C/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions
    • C12Q1/6853Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855Ligating adaptors
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/975Kit

Abstract

Methods of synthesizing multiple copies of a target nucleic acid sequence autocatalytically under conditions of substantially constant temperature, ionic strength, and pH are provided in which multiple RNA copies of the target sequence autocatalytically generate additional copies.
These methods are useful for generating copies of a nucleic acid target sequence for purposes which include assays to quantitate specific nucleic acid sequences in clinical, environmental, forensic and similar samples, cloning and generating probes.

Description

DESCRIPTION
Nucleic Acid Sequence Amtilifica »n Method Field of the Invention This invention relates to methods for increasing the number of copies of a specific nucleic acid sequence or "target sequence" which may be present either alone or as a component, large or ;small, of a homogeneous or hetero-geneous mixture of nucleic acids. The mixture of nucleic IO acids may be that found in a sample taken for diagnostic testing, environmental testing, for research studies, far the preparation of reagents or materials for other processes such as cloning, or for other purposes.
The selective amplification of specific nucleic acid sequences is of value in increasing the sensitivity of diagnostic and environmental assays while maintaining specificity; increasing the sensitivity, convenience, accuracy and reliability of a variety of research procedures; and providing ample supplies of specific 2~ oligonucleotides for various purposes.
The present invention is particularly suitable for use in environmental and diagnostic testing due to the convenience with which it may be practiced.
2'S Background of the Invention The detection and/or quantitation of specific nucleic acid sequences is an increasingly important technique for identifying and classifying microorganisms, diagnosing infectious diseases, detecting and characterizing genetic 3~) abnormalities, identifying genetic changes associated with cancer, studying genetic susceptibility to disease, and measuring response to various types of treatment. Such procedures have also foLUnd expanding uses in detecting and ~O
c, r ~., ,a ~ i ~'g~ . ~~ ri quantitating microorganisms in foodstuffs, environmental samples, seed stocks, and other types of material where the presence of specific microorganisms may need to be monitored. Other applications are found in the forensic sciences, anthropology, archaeology, and biology where measurement of the relatedness of nucleic acid sequences has been used to identify criminal suspects, resolve paternity disputes, construct genealogical and phylogenetic trees, and aid in classifying a variety of 1.0 life forms.
A common method for detecting and quantitating specific nucleic acid sequences is nucleic acid hybridization. This method is based on the ability of two nucleic acid strands which contain complementary or essentially complementary sequences to specifically associate, under appropriate conditions, to form a double-stranded structure. To detect and/or quantitate a specific nucleic acid sequence (known as the "target sequence"), a labelled oligonucleotide (known as a "probe") is prepared which contains sequences complementary to those of the target sequence. The probe is mixed with a sample suspected of containing the target sequence, and conditions suitable for hybrid formation are created. The probe hybridizes to the target sequence if it is present in the sample. The probe-target hybrids axe then separated from the single-stranded probe in one of a variety of ways. The amount of label associated with the hybrids is measured.
The sensitivity of nucleic acid hybridization assays is limited primarily by the specific activity of the probe, the rate and extent of the hybridization reaction, the performance of the method for separating hybridized and unhybridized probe, and the sensitivity with which the label can be detected. Under the best conditions, direct hybridization methods such as that described above can detect about 1 x 105 to 1 x 1.06 target molecules . The mast sensitive procedures may lack many of the features required for routine clinical and environmental testing such as speed, convenience, and economy. Furthermore, their sensitivities may not be sufficient for many desired applications. Infectious diseases may be associated with _°> as few as one pathogenic microorganism per 10 ml of blood or other specimen. Forensic investigators may have available only trace amounts of tissue available from a crime scene. Researchers may need to detect and/or quan-titate a specific gene ;sequence that is present as only a 1t1 tiny fraction of all the sequences present in an organism's genetic material or in the messenger RNA
population of a group of cells.
As a result of the interactions among the various components and component steps of this type of assay, 1°_. there is almost always an inverse relationship between sensitivity and specificity. Thus, steps taken to increase the sensitivit_~r of the assay (such as increasing the specific activity of: the probe) may result in a higher percentage of false positive test results. The linkage 2C~ between sensitivity and; specificity has been a signifi-cant barrier to improving the sensitivity of hybridization assays. One solution to this problem would be to specifically increase the amount of target sequence present using an amplification procedure. Amplification 25 of a unique portion of the target sequence without requiring amplification of a significant portion of the information encoded in the remaining sequences of the sample could give an increase in sensitivity while at the same time not compromising specificity. For example, a 30 nucleic acid sequence of 25 bases in length has a probability of occurring by chance of 1 in 4ZS or 1 in 10~s since each of the 25 positions in the sequence may be occupied by one of four different nucleotides.
A method for specifically amplifying nucleic acid 35 sequences termed the "polymerase chain reaction" or "PCR"
has been described by Mullis et al. (See U.S. patents 4,683,195, 4,683,202 and 4,800,159 and ~et~ods in Enzvmoloav, Volume 155, 1987, pp, 335-350).
The procedure uses repeated cycles of primer-dependent nucleic acid synthesis occurring simultaneously using each strand of a complementary sequence as a template. The sequence which is amplified is defined by the locations of the primer molecules that initiate synthesis. The primers are complementary to the 3'-terminal portion of the target sequence or its complement and must complex with those 1~) sites in order for nuclsaic acid synthesis to begin. After extension product synthesis, the strands are separated, generally by thermal denaturation, before the next synthesis step. In the PCR procedure, copies of both strands of a complementary sequence are synthesized.
1-''> The strand separation step used in PCR to separate the newly synthesized strands at the conclusion of each cycle of the PCR reaction is often thermal denaturation.
As a result, either a thermostable enzyme is required or new enzyme must be added between thermal denaturation 2C~ steps and the initiation of the next cycle of DNA
synthesis. The requirement of repeated cycling of reaction temperature between several different and extreme temperatures is a disadvantage of~the PCR procedure. In order to make the PCR convenient, expensive programmable 25 thermal cycling instruments are required.
The PCR procedure has been coupled to RNA
transcription by incorporating a promoter sequence into one of the primers used in the PCR reaction and then, after amplification by the PCR procedure for several 30 cycles, using the double:-stranded DNA as template for the transcription of single-stranded RNA. (See, e-a. Murakawa et al., DNA 7:287-295 (1.988).
Other methods for amplification of a specific nucleic acid sequence comprise a series of primer hybridization, 35 extending and denaturing steps to provide an intermediate double stranded DNA molecule containing a promoter sequence through the use of a primer. The double stranded ;j :s DNA is used to produce multiple RNA copies of the target sequence. The resulting RNA copies can be used as target sequences to produce further copies and multiple cycles can be performed. (See, ewe., Burg, et al., WO 89/1050 5 and Gingeras, et al., WO 88/10315.) Methods far chemically synthesizing relatively large amounts of DNA of a specified sequence in vitro are well known to those skilled in the art; production of DNA in this way is now commonplace. However, these procedures 1o are time°consuming and cannot be easily used to synthesize oligonucleotides much greater in length than about 100 bases. Also, the entire base sequence of the DNA to be synthesized must be known. These methods require an expensive instrument capable of synthesizing only a single sequence at one time. .Operation of this instrument requires considerable training and expertise. Methods for the chemical synthesis of RNA have been more difficult to develop.
Nucleic acids may be synthesized by techniques which involve cloning or insertion of specific nucleic acid sequences into the genetic material of microorganisms so that the inserted sequences are replicated when the organism replicates. If the sequences are inserted next to and downstream from a suitable promoter sequence, RNA
copies of the sequence or protein products encoded by the sequence may be produced. Although cloning allows the production of virtually unlimited amounts of specific nucleic acid sequences, due to the number of manipula°
tions involved it may not be suitable for use in diagnostic, environmental, or forensic testing. Use of cloning techniques requires considerable training and expertise. The cloning of a single sequence may consume several man°months of effort or more.
Relatively large amounts of certain RNAs may be made using a recombinant single-stranded-RNA molecule having a recognition sequence for the binding of an RNA°directed polymerase, preferably ~,0 replicase. (See, e.a., U.S.

~ ~, ,'.~ :i ~
Patent No. 4,786,600 to Kramer, et al.) A number of steps are required to insert the specific sequence into a DNA
copy of the variant molecule, clone it into an expression vector, transcribe it into RNA and then replicate it with ~a replicase.
Summary of the Invention The present invention is directed to novel methods of synthesizing multiple copies of a target nucleic acid sequence which are autocatalytic (i.e., able to cycle automatically without the need to modify reaction conditions such as temperature, pH, or ionic strength and using the product of one cycle in the next one).
The present method includes (a) treating an RNA
target sequence with a first oligonucleotide which comprises a first primer which has a complexing sequence sufficiently complementary to the 3'-terminal portion of the target to complex therewith and which optionally has a sequence 5' to the priming sequence which includes a promoter for an RNA polymerase under conditions whereby an oligonucleotide/target sequence complex is formed and DNA
synthesis may be initiated, (b) extending the first primer in an extension reaction using the target as a template to give a f first DNA primer extension product complementary to the RNA target, (c) separating the DNA extension product from the RNA target using an enzyme which selectively degrades the RNA target; (d) treating the DNA primer extension product with a second oligonualeotide which comprises a primer or a splice template and which has a complexing sequence sufficiently complementary to the 3'-terminal portion of the DNA primer extension product to complex therewith under conditions whereby an oligonucleotide/ target sequence complex is formed and DNA
synthesis may be initiated, provided that if the first oligonucleotide does not have a promoter, then the second oligonucleotide is a splice template which has a sequence 5' to the complexing sequence which includes a promoter r e' ~~- ~h i v fiY 1 J ',!
for an RNA polymerise; (e) extending the 3'-terminus of either the second oligonucleotide or the first primer extension product, or both, in a DNA extension reaction to produce a template for the RNA polymerise; and (f) using the template to produce multiple RNA copies of the target sequence using an RNA polymerise which recognizes the promoter sequence. The oligonucleotide and RNA copies may be used to autocatalytically synthesize multiple copies of the target sequence.
Tn one aspect of the present invention, the general method includes (a) treating an RNA target sequence with a first oligonucleotide which comprises a first primer which has a complexing sequence sufficiently complementary to the 3'-terminal portion of the target to complex there-with and which has a sequence 5' to the complexing sequence which includes a promoter for an RNA polymerise under conditions whereby an oligonucleotide/target complex is formed and DNA synthesis may be initiated, (b) extend-ing the first primer in an extension reaction using the target as a template to give a first DNA primer extension product complementary to the RNA target, (c) separating the first DNA primer extension product from the RNA target using an enzyme which selectively degrades the RNA target;
(d) treating the DNA primer extension product with a second oligonucleotide which comprises a second primer which has a complexing sequence sufficiently complementary to the 3'-terminal portion of the DNA primer extension product to complex therewith under conditions whereby an oligonucleotide/target complex is formed and DNA synthesis may be initiated; (e) extending the 3'-terminus of the second primer in a DNA extension reaction to give a second DNA primer extension product, thereby producing a template for the RNA polymerise; and (f) using the template to produce multiple RNA copies of the target sequence using an RNA polymerise which recognizes the promoter sequence.
The oligonucleotide and RNA copies may be used to autocatalytically synthesize multiple copies of the target r 2~2~~~8 sequence. This aspect further includes: (g) treating an RNA copy from step (f) with the second primer under conditions whereby an oligonucleotide/ target sequencA
complex is formed arid DNA synthesis may be initiated; (h) extending the 3' terminus of the second primer in a DNA
extension reaction to give a second DNA primer extension product using the RNA copy as a template; (i) separating the second DNA primer extension product from the RNA copy using an enzyme which selectively degrades the RNA copy;
(j) treating the second DNA primer extension product with the first primer under conditions whereby an oligonucleotide/target sequence complex is formed and DNA
synthesis may be initiated; (k) extending the 3' terminus of the second primer extension product in a DNA extension reaction to produce a template for an RNA polymerase; and (1) using the 'template of step (k) to produce multiple copies of the target sequence using an RNA polymerase which recognizes the promoter. Using the RNA copies of step (1), steps (g) to (k) may be autocatalytically repeated to synthesize multiple copies of the target sequence. The first primer which in step (k) acts as a splice template may also be extended in the DNA extension reaction of step (k).
Another aspect of the general method of the present invention provides a method which comprises (a) treating an RNA target sequence with a first primer which has a complexing sequence sufficiently complementary to the 3' terminal portion of the target sequence to complex therewith under conditions whereby an oligonucleotide/target sequence complex is formed and DNA
synthesis may be initiated; (b) extending the 3' terminus of the primer in an extension reaction using the target as a template to give a DNA primer extension product complementary to the RNA target; (c) separating the DNA
extension product from the RNA target using an enzyme which selectively degrades the RNA target; (d) treating the DNA primer extension product with a second oligonucleotide which comprises a splice template which has a complexing sequence sufficiently complementary to the 3°-terminus of the primer extension product to complex therewith and a sequence 5' to the complexing sequence which includes a promoter for an RNA polymerase under conditions whereby an oligonucleotide/target sequence complex is formed and DNA synthesis may be initiated; (e) extending the 3° terminus of the DNA primer extension product to add thereto a sequence complementary to the promoter, thereby producing a template for an RNA poly-merase; (f) using the template to produce multiple RNA
copies of the target sequence using an RNA polymerase which recognizes the promoter sequence; and (g) using the RNA copies of step (f), autacatalytically repeating steps (a) to {f) to amplify the target sequence. Optionally, the splice template of step (d) may also function as a primer and in step (e) be extended to give a second primer extension product using the first primer extension product as a template.
Tn addition, in another aspect of the present .
invention, where the sequence sought to be amplified is present as DNA, use of an appropriate Preliminary Procedure generates RNA copies which may then be amplified according to the General Method of the present invention.
Accordingly, in another aspect, the present invention is directed to Preliminary Procedures far use in conjunction with the amplification method of the present invention which not only can increase the number of copies present to be amplified, but also can provide RNA copies of a DNA sequence for amplification.
The present invention is directed to methods for increasing the number of copies of a specific target nucleic acid sequence in a sample. In one aspect, the present invention involves cooperative action of a DNA
polymerase {such as a reverse transcriptase) and a DNA-dependent RNA polymerase (transcriptase) with an enzymatic hybrid-separation step to produce products that may them-l0 selves be used to produce additional product, thus resulting in an autocaitalytic reaction without requiring manipulation of reaction conditions such as thermal cycling. In some embodiments of the methods of the present invention which include a Preliminary Procedure, all but the initial steps) of the preliminary procedure are carried out at one temperature.
The methods of the: present invention may be used as a component of assays to detect and/or quantitate specific io nucleic acid target sequences in clinical, environmental, forensic, and similar samples or to produce large numbers of copies of DNA and/or RNA of specific target sequence for a variety of uses. These methods may also be used to produce multiple DNA copies of a DNA target sequence for cloning or to generate probes or to produce RNA and DNA
copies for sequencing.
In one example of a typical assay, a sample to be amplified is mixed with a buffer concentrate containing the buffer, salts, magnesium, nucleotide triphosphates, 2~D primers and/or splice templates,~'dithiothreitol, and spermidine. The reaction is then optionally incubated near 100°C for two m~.nutes to denature any secondary structure. After coo7ling to room temperature, if the target is a DNA target without a defined 3' terminus, 2'S reverse transcriptase is added and the reaction mixture is incubated for 12 minutes at 42°C. The reaction is again denatured near 100°C, this time to separate the primer extension product from the DNA template. After cooling, reverse transcriptase, RNA polymerise, and RNAse 3~7 H are added and the reaction is incubated for two to four hours at 37°C. The reaction can then be assayed by dena-turing the product, adding a probe solution, incubating 20 minutes at 60°C, adding a solution to selectively hydrolyze the unhybridized probe, incubating the reaction 3!5 six minutes at 60°C, and measuring the remaining chemiluminescence in a luminometer. (See, e.a., Arnold, ~t ~~. WO 89/02476.) This typical assay method is referred to as "HPA"). The products of the methods of the present invention may be used in many other assay systems known to those skilled in the art..
If the target has a defined 3' terminus or the target is RNA, a typical assay includes mixing the target with the buffer concentrate mentioned above and denaturing any secondary structure. After cooling, reverse trans-criptase, RNA polymerase, and RNAse H are added and the mixture is incubated for' two to four hours at 37°C. The reaction can then be assayed as described above.
The methods of t:he present invention and the materials used therein may be incorporated as part of diagnostic kits for use in diagnostic procedures.
Definitions As used herein, the following terms have the following meanings unless expressly stated to the contrary.
1. Template A "template" is a nucleic acid molecule that is being copied by a nucleic acid polymerase. A template may be either single-stranded, double-stranded or partially double-stranded, depending on the polymerase. The synthesized copy is complementary to the template or to at least one strand of a doulble-stranded or partially double-stranded template. Both. RNA and DNA are always synthe-sized in the 5' to 3' direction and the two strands of a nucleic acid duplex always are aligned so that the 5' ends of the two strands are at opposite ends of the duplex (and, by necessity, so then are the 3' ends).

'~0~0~~8 2. Primer, Splice Template A "primer" is an oligonucleotide that is comple-mentary to a template which complexes (by hydrogen bonding or hybridization) with the template to give a primer/
template complex for initiation of synthesis by a DNA
polymerise, and which is extended by the addition of covalently bonded bases linked at its 3' end which are complementary to the template in the process of DNA
synthesis. The result is a primer extension product.
Virtually all DNA polymerises (including reverse transcriptases) that are known require complexing of an oligonucleotide to a single-stranded template ("priming") to initiate DNA synthesis, whereas RNA replication and transcription (copying of RNA from DNA) generally do not require a primer. Under appropriate circumstances, a primer may act as a splice template as well (see definition of '°splice template" 'that follows).
A "splice template" is an oligonucleotide that complexes with a single-stranded nucleic acid and is used as a template to extend the 3' terminus of a target nucleic acid to add a specific sequence. The splice template is sufficiently complementary to the 3' terminus of the target nucleic acid molecule, which is to be extended, to complex therewith. A DNA- or RNA-dependent DNA polymerise is then used to extend the target nucleic acid molecule using the sequence 5' to the complementary region of the splice template as a template. The exten-sion product of the extended molecule has the specific sequence at its 3'-terminus which is complementary to the sequence at the 5'-terminus of the splice template.
If the 3' terminus of the splice template is not blocked and is complementary to the target nucleic acid, it may also act as a primer and be extended by the DNA
polymerise using the target nucleic acid molecule as a template. The 3' terminus of the splice template can be blocked in a variety of ways, including having a 3'-terminal dideoxynucleotide or a 3'-terminal sequence non-'~0~0~~'~

ccmplementary to the target, ar in other ways well known to those skilled in the art.
Either a primer or a splice template may complex with a single-stranded nucleic acid and serve a priming function for a DNA polymerise.
3. Target Nucleic Acidt Target Sequence A '°target nucleic acid" has a "target sequence" to be amplified, and may be either single-stranded ar double stranded and may include other sequences besides the target sequence which may not be amplified.
The term "target sequence" refers to the particular nucleotide sequence of the target nucleic acid which is to be amplified. 'fhe °°target sequence" includes the camplexing sequences to which the oligonucleotides (primers and/or splice template) complex during the processes of the present invention. Where the target nucleic acid is originally single-stranded, the term "target sequence" will also refer to the sequence comple-mentary to the °'target sequence" as present in the target nucleic acid. Where the "target nucleic acid" is originally double-stranded, the term "target sequence"
refers to both the (+) and (-) strands.
4. Promoter/Prometer Sequence A °promoter sequence" is a specific nucleic acid sequence that is recognized by a DNA-dependent RNA
polymerise ( "transcriptase" ) as a signal to bind to the nucleic acid and begin the transcription of RNA at a specific site. For binding, such transcriptases generally require DNA which is double--stranded in the portion comprising the promoter sequence and its complement; the template portion (sequence to be transcribed) need not be double-stranded. Individual DNA-dependent RNA polymerises recognize a variety of different promoter sequences which can vary markedly in their efficiency in promoting tran-scription. When an RNA polymerise binds to a promoter sequence to initiate transcription, that promoter sequence is not part of the sequence transcribed. Thus, the RNA
transcripts produced thereby will not include that sequence.
5. ANA-dependent ~'.t~$ ~g~~merase A "DNA-dependent DNA polymexase" is an enzyme that synthesizes a complementary DNA copy from a DNA template.
Examples are DNA polymerise T from ~. coli and bacteriophage T7 DNA polymerise. A11 known DNA-dependent DNA polymerises require a complementary primer to initiate synthesis. It is known that under suitable conditions a DNA-dependent DNA polymerise may synthesize a complementary DNA copy from an RNA template.
6. DNA-dependent RICA Polymerise (Transcriptasel A '°DNA-dependent RNA polymerise" or "transcriptase"
is an enzyme that synthesizes multiple RNA copies from a double-stranded or partially-double stranded DNA molecule having a (usually double-stranded) promoter sequence. The .
RNA molecules ("transcripts") are synthesized in the 5' -3' direction beginning at a specific pasition just downstream of the promoter. Examples of transcriptases are the DNA-dependent RNA polymerise from ~: coli and bacteriophages T7, T3, and SP6.
7, RNA-dependent DNA polymerise (Reverse Transcri~tase) An '°RNA-dependent DNA polymerise" or '°reverse transcriptase°' is an enzyme that synthesizes a complementary DNA copy from an RNA template. All known reverse transcriptases also have the ability to make a complementary DNA copy from a DNA template; thus, they are both RNA- and DNA-dependent DNA polymerises. A primer is required to initiate synthesis with both RNA and DNA templates.

8. RNAse H
An "RNAse H" is an enzyme that degrades the RNA
portion of an RNA:DNA duplex. RNAse H's may be endonucleases or exonucleases. Most reverse transcriptase enzymes normally cantain an RNAse H activity in addition to their polymerise activity. However, other sources of the RNAse H are available without an associated polymerise activity. The degradation may result in separation of RNA
from a RNA:DNA complex. Alternatively, the RNAse H may simply cut the RNA at various locations such that portions of the RNA melt off or permit enzymes to unwind portions of the RNA.
9. Plus/Minus Strand~s~
Discussions of nucleic acid synthesis are greatly simplified and clarified by adopting terms to name the two complementary strands of a nucleic acid duplex.
Traditionally, the strand encoding the sequences used to produce proteins or structural RNAs was designated as the "plus°' strand and its complement the "minus" strand. It is now known that in many oases, both strands are functional, and the assignment of the designation °'plus"
to one and "minus',' to the other must then be arbitrary.
Nevertheless, the terms are very useful for designating the sequence orientation of nucleic acids and will be employed herein for that purpose.
10. Hybridize,, Hybridization The terms ''hybridize" and "hybridization° refer to the formation of complexes between nucleotide sequences which are sufficiently complementary to form complexes via Watson-Crick base pairing. Where a primer (ar splice template) °'hybridizes" with target (template), such complexes (or hybrids) are sufficiently stable to serve the priming function required by the DNA polymerise to initiate DNA synthesis.

~a~aa~~
11. Primer sequences The sequences of the primers referred to herein are set Earth below.
I3BV region 2 primers (+): 5'CACCAAATGCCCCTATCTTATCAACACTTCCGG3' (-): 5'AATTTAATACGACTCACTATAGGGAGACCCGAGATTGAG
ATCTTCTGCGAC3' Prabe (+): 5'GGTCCCCTAGAAGAAGAACTCCCTCG3' HIV region 1 primers (+): 5'AATTTAATACGACTCACTATAGGGAGACAAGGGACTTTCC
GCTGGGGACTTTCC3' 5'GTCTAACCAGAGAGACCCAGTACAGGC3' Probe sequence:
5'GCAGCTGCTTATATGCAGGATGTGAGGG3' HIV region 2 primers (+): 5'AATTTAATACGACTCACTATAGGGAGACAAATGGCA
GTATTCATCCACA3' (-) : 5' CCC'1.'TCACC~fTTCCAGAG3' Probe sequence:
(-): 5'CTACT.ATTCTTTCCCCTGCACTGTACCCC3' HIV region 3 primers (+): 5'CTCGACGCAGGACTCGGCTTGCTG3' (-): 5'AATTTAATACGACTCACTATAGGGAGACTCCCCCGCTT
AATACTGACGCT3' Probe:
(+): 5'GACTAGCGGAGGCTAGAAGGAGAGAGATGGG3' HIV region 4 primers (+): 5'AATTTAATACGACTCACTATAGGGAGAGACCATCAATGAGGAA
GCTGCAGAATG3' (-): 5°CCATCCTATTTGTTCCTGAAGGGTAC3°
Probe:
(-): 5'CTTCCCCTTGGTTCTGTCATCTGGCC3' HIV region 5 primers (+): 5'GGCAAATGGTACATCAGGCCATATCACCTAG3' (-): 5'AATTTAATACGACTCACTATAGGGAGAGGGGTGGCTCCTT
CTGATAATGCTG3' Probe:
5°GAAGGCTTTCAGCCCAGAAGTAATACCCATG3' BCL-2 chromosomal translocation major breakpoint t(14;18) primers (-): 5'GAATTAATACGACTCAC'I'ATAGGGAGACCTGAGGAGACGGTGACC3' (+): 5 "rATGGTGGTTTGACCTTTAG3' Probes:
5'GGCTTTCTCATGGCTGTCCTTCAG3' 5°GGTCT'rCCTGAAATGCAGTGGTCG3' CML chromosomal translocation t(9;22) primers (--): 5'GAATTAATACGACTCACTATAGGGAGACTCAGAC
CCTGAGGCTCAAAGTC3' (+): 5°GGAGCTGCAGATGCTGACCAAC3°
Probe:
5'GCAGAGTTCAAAAGCCCTTCAGCGG3' 12 . S~ecif icity Characteristic of a nucleic acid sequence which describes its 'ability to distinguish between target and non-target sequences dependent on sequence and assay conditions.
Brief Descr ~tion of the Drawing FIGS. 1A to 10 depict the General Methods of the present invention.
FIGS. 2A to 2E depict the embodiment of the present invention referred to as Preliminary Procedure I.
FIG. 3 depicts the embodiment of the present invention referred to as Preliminary Procedure II.
FIG s.4A to 4D depicts the improved amplification method.
FIG. 5 shows the results of experiments testing the hypothesis that RNAse H from AMV and MMLV and E. coli have specific RNA cleavage sites.
FIG. 6 shows the results of incorporation of 3zP-labeled primers during amplification.

Detailed Description of the Invention In accordance with the present invention, novel methods and compositions are provided for the amplification of specific nucleic acid target sequences for use in assays for the detection and/or quantitation of specific nucleic acid target sequences or for the produc-tion of large numbers of copies of DNA and/or RNA of specific target sequences for a variety of uses.
I. General Method In a preferred aspect, the present invention provides an autacatalytic amplification method which synthesizes large numbers of DNA and RNA copies of an RNA target sequence. The target nucleic acid contains the target sequence to be amplified. The target sequence is that region of the target nucleic acid which is defined on either end by the primers, splice templates, and/or the natural target nucleic acid termini and includes both the (+) and (-) strands.
In one aspect, this method comprises treating a target nucleic acid comprising an RNA target sequence with a first oligonucleotide which comprises a first primer which has a c:omplexing sequence sufficiently complementary to the 3'-terminal portion o~ the target sequence to complex therewith and which optionally has a sequence 5' to the camplexing sequence which includes a promoter sequence for an RNA polymerase under conditions whereby an aliganucleatide/target sequence complex is formed and DNA synthesis may be initiated. The first oliganucleo-tide primer may also have other sequences 5' to the priming sequence. The 3'-end of the first primer is extended by an appropriate DNA polymerase in an extension reaction using the RNA as a template to give a first DNA
primer extension product which is complementary to the RNA
template. The first primer extension product is separated (at least partially) from the RNA template using an enzyme which selectively degrades the RNA template. Suitable 202~~5g enzymes axe those which selectively act on the RNA strand of an RNA-DNA complex and include enzymes which comprise an RNAse H. Although some reverse transcriptases include an RNAse H activity, it may be preferable to add exo-genous RNAse H, such as an ~, coli RNAse H.
The single-stranded first primer extension product is treated with a second oligonucleotide which comprises a second primer or a splice template which has a complexing sequence sufficiently complementary to the 3°-terminal portion of target sequence contained in the first primer extension product to complex therewith, under conditions whereby an oligonucleotide/ target sequence complex is formed and DNA synthesis may be initiated. If the first primer does not have a promoter then the second oligo-nucleotide is a splice template which has a sequence 5' to the complexing region which includes a promoter fox an RNA
polymerase. Optionally, the splice template may be blocked at its 3' terminus. The 3' terminus of the second oligonucleotide and/or the primer extension product is 2o extended in a DNA extension reaction to produce a template far a RNA polymerase. The RNA copies or transcripts produced may autocatalytically multiply without further manipulation, Where an oligonucleotide functions as a splice template, its primer function is not required. Thus, the 3' terminus of the splice template may be either blocked or unblocked. The components of the resulting reaction mixture (i.e., an RNA target which allows production of a first primer extension product with a defined 3' terminus, 3o a first primer, and a splice template either blocked or unblocked) function to autocatalytically synthesize large quantities of RNA and DNA.
In one aspect of the present invention, the first and second oligomers both are primers. The first primer has a sequence 5' to the camplexing sequence which includes a promoter fox a RNA polymerase and may include other sequences. The second primer may also include a sequence 2'~ ~'~ 9 ~ ~
5' to the complexing sequence which may include a promoter for an RNA polymerise and optionally other sequences.
Where both primers have a promoter sequence, it is preferred that both sequences are recognized by the same 5 RNA polymerise unless it is intended to introduce the second promoter for other purposes, such as cloning. The 3'-end of the second primer is extended by an appropriate DNA polymerise in an extension reactian to produce a second DNA primer extension product complementary to the 10 first primer extension product. Note that as the first primer defined one end of the target sequence, the second primer now defines the other end. The double-stranded product of the second extension reaction is a suitable template for the production of RNA by an RNA polymerise.
15 If the second primer also has a promoter sequence, transcripts complementary to both strands of the double-stranded template will be produced during the autocata-lytic reaction. The RNA transcripts may now have different termini than the target nucleic acid, but the 20 sequence between the first primer and the second primer remains intact. The RNA transcripts so produced may automatically recycle in the above system without further manipulation. Thus, this reaction is autocatalytic.
If the comnlexina seauence of the second primer complexes with the 3' terminus of the first primer extension product, the second primer may act as a splice template and the first primer extension product may be extended to add any sequence of the second primer 5' to the priming sequence to the first primer extension product. (See, e.g., Figures 1E and 1G) If the second primer acts as a splice template and includes a promoter sequence 5' to the complexing sequence, extension of the first primer extension product to add the promoter sequence produces an additional template for an RNA
polymerise which may be transcribed to produce RNA copies of either strand. (See Figures lE and 1G) Inclusion of promoters in both primers may enhance the number of copies of the target sequence synthesized.
Another aspect of the general method of the present invention includes using a first oligonucleotide which comprises a primer and a second oligonucleotide which comprises a splice template and which may or may not be capable of acting as a primer per se ( in that it is not itself extended in a primer extension reaction). This aspect of the general method comprises treating a target nucleic acid comprising an RNA target sequence with a first oligonucleotide primer which has a complexing sequence sufficiently complementary to the 3' terminal portion of the target sequence to complex therewith under conditions whereby an oligonucleotide/target sequence 1.5 complex is formed and DNA synthesis may be initiated. The first primer may have other sequences 5' to the complexing sequence, including a promoter. The 3' end of the first primer is extended by an appropriate DNA polymerase in an extension reaction using the RNA as a template to give a first primer extension product which is complementary to the RNA template. The first primer extension product is separated from the RNA template using an enzyme which selectively degrades the RNA template. Suitable enzymes are those which selectively act on the RNA strand of an RNA-DNA complex and include enzymes which comprise an RNAse H activity. Although some reverse transcriptases include an RNase H activity, it may be preferable to add exogenous RNAse H, such as an E. calf RNAse H. The single stranded first primer extension product is treated with a splice template which has a complexing sequence suffi-ciently complementary to the 3'-terminus of the primer extension product to complex therewith and a sequence 5' to the complexing sequence which includes a promoter for an RNA polymerase under conditions whereby an oligonucleotide/target sequence complex is formed and DNA
synthesis may be initiated. The 3' terminus of the splice template may be either blocked (such as by addition of a dideoxynucleotide) or uncomplimentary to the target nucleic acid (so that it does not function as a primer) or alternatively unblocked. The 3' terminus of the first primer extension product is extended using an appropriate DNA polymerise in a DNA extension reaction to add to the 3' terminus of the first primer extension product a sequence complementary to the sequence of the splice template 5' to the complexing sequence which includes the promoter. If the 3' terminus is unblocked, the splice template may be extended to give a second primer extension product complementary to the first primer extension product. The product of the extension reaction with the splice template (whether blocked or unblocked) can function as a template far RNA synthesis using an RNA
polymerise which recognizes the promoter. As noted above, RNA transcripts so produced may automatically recycle in the above system without further manipulation. Thus, the reaction is autocatalytic.
In some embodiments, the target sequence to be amplified is defined at both ends by the lacation of specific sequences complementary to the primers (or splice templates) employed. In other embodiments, the target sequence is defined at one location of a specific sequence, complementary to a primer molecule employed and, at the opposite end, by the location of a specific sequence that is cut by a specific restriction endo-nuclease, or by other suitable means, which may include a natural 3' terminus. In other embodiments, the target sequence is defined at both ands by the location of specific sequences that are cut by one or more specific restriction endonuclease(s).
In a preferred embodiment of the present invention, the RNA target sequence is determined and then analyzed to determine where RNAse H degradation will cause cuts or removal of sections of RNA from the duplex. Analyses can be conducted to determine the effect of the RNAse degradation of the target sequence by RNAse H present in ~o~o~~~

AMV reverse transcriptase and MMLV reverse transcriptase, by E. coli RNAse H or other sources and by combinations thereof .
In selecting a primer set, it is preferable that one of the primers be selected so that it will hybridize to a section of RNA which is substantially nondegraded by the RNAse H present in the reaction mixture. If there is substantial degradation, the cuts in the RNA strand in the region of the primer may inhibit initiation of DNA
1o synthesis and prevent extension of the primer. Thus, it is preferred to select a primer which will hybridize with a sequence of the RNA target, located so that when the RNA
is subjected to RNAse H, there is no substantial degradation which would prevent formation of the primer extension product.
The site for hybridization of the promoter-primer is chosen so that sufficient degradation of the RNA strand occurs to permit removal of the portion of the RNA strand hybridized to the portion of the DNA strand to which the promoter-primer will hybridize. Typically, only portions of RNA are removed from the RNA: DNA duplex through RNAse H degradation and a substantial part of the RNA strand remains in the duplex.
Formation of the promoter-.containing double stranded product for RNA synthesis is illustrated in Figure 4. As illustrated in Figure 4, the target RNA strand hybridizes to a primer which is selected to hybridize with a region of the RNA strand which is not substantially degraded by RNAse H present in the reaction mixture. The primer is then extended to form a DNA strand complementary to the RNA strand. Thereafter, the RNA strand is cut or degraded at various locations by the RNAse H present in the reaction mixture. It is to be understood that this cutting or degradation can occur at this point or at other times during the course of the reaction. Then the RNA
fragments dissociate from the DNA strand in regions where significant cuts or degradation occur. The promoter-primer then hybridizes to the DNA strand at its 3' end, where the RNA strand has been substantially degraded and separated from the DNA strand. Next, the DNA strand is extended to form a double strand DNA promoter sequence, thus forming a template for RNA synthesis. It can be seen that this template contains a double-stranded DNA promoter sequence. When this template is treated with RNA
polymerase, multiple strands of RNA are formed.
Although the exact nature of the RNA degradation resulting from the RNAse H is not known, it has been shown that the result of RNAse H degradation on the RNA strand of an RNA: DNA hybrid resulted in dissociation of small pieces of RNA from the hybrid. It has also been shown that promoter-primers can be selected which will bind to the DNA after RNAse H degradation at the area where the small fragments are removed.
Figures 1 and 2, as drawn, do not show the RNA which may remain after RNAse H degradation. It is to be understood that although these figures generally show complete removal of RNA from the DNA:RNA duplex, under the preferred conditions only partial removal occurs as illustrated in Figure 3. By reference to Figure lA, it can be seen that the proposed mechanism may not occur if a substantial portion of the RNA strand of Figure 1 remains undegraded thus preventing hybridization of the second primer or extension of the hybridized second primer to produce a DNA strand complementary to the promoter sequence. However, based upon the principles of synthesis discovered and disclosed in this application, routine modifications can be made by those skilled in the art according to the teachings of this invention to provide an effective and efficient procedure for amplification of RNA.
As may be seen from the descriptions herein and Figures 1A to 1Q, the method of the present invention embraces optional variations.

2~2~~~~
Figure lA depicts a method according to the present invention wherein the target nucleic acid has additional sequences 3' to the target sequence. The first oligonucleotide comprises a first primer having a promoter 5 5' to its complexing sequence which camplexes with the 3' terminal portion of the target sequence of a target nucleic acid (RNA) which has additional sequences 3' to the end of the target sequence. The second oligonucleotide comprises a second primer which complexes 10 with the 3° terminal portion of the first primer extension product, coinciding with. the 3' terminus of the first primer extension product. In step (1), the first primer does not act as a splice template due to the additional sequences 3' to the target sequence; however, in step 15 (ZO), the first primer can act as a splice template, since the second primer extension product does not have additional sequences 3° to the target sequence.
Figure 1B depicts a method according to the present invention wherein the target nucleic acid (RNA) has 20 additional sequences both 5' and 3' to the target sequence. The first oligonucleotide is as depicted in Figure lA. The second oligonucleotide comprises a primer which complexes to the 3° terminal portion of the target sequence of the first primer extension product which has 25 additional sequences 3' to the target sequence.
Figure 1C depicts a target nucleic acid (RNA) which.
has defined 5' and 3' ends and, thus, has no additional sequences either 5° or 3° to the target sequence. The first oligonucleotide is as depicted in Figures lA and 1B, but since it complexes with the 3° terminus of the target nucleic acid, it acts as both a primer and splice template in Step 1. The second oligonucleotide is as depicted in Figure 1A.
Figure 1D depicts a target nucleic acid (RNA) having a defined 3° end and, thus, has no additional sequences 3' to the target sequence, but does have additional sequences 5' to the target sequence. The first oligonucleotide is as depicted in Figure 1C and functions as both a primer and a splice template. The second oligonucleotide is as depicted in Figure 1B, Figure lE depicts a target nucleic acid (RNA) which has a defined 5' end but which has additional sequences 3' to the target sequence. The first oligonucleotide is as depicted in Figure lA. The second oligonucleotide comprises a second primer which, since it complexes with the 3°-terminus of the first primer extension product, to also comprises a splice template. The second oligo--nucleotide also has a promoter 5' to its complexing sequence.

Figure 1F depicts a target nucleic acid (RNA) having additional sequences both 5' and 3' to the target 15sequence. The first oligonucleotide is as depicted in Figure 1A. The second oligonucleotide is as depicted in Figure 1E, except it cannot act as a splice template in step (~), since the first primer extension product has additional sequences 3' to the target sequence.

20Figure 1G depicts a target nucleic acid (RNA) which has both defined 5' and 3' ends, having no sequences besides target sequence. The first oligonucleotide the is as depictedin Figure 1C and the second oligonucleotide as depicted Figure lE. Since the target has no additional in 25sequences, both oligonucleotides also act as splice templates.

Figure 1H depicts a target nucleic acid (RNA) which has a defined 3 end, having no sequences 3' to the target sequence, ut has additional sequences 5' to the b target 30sequence. The first oligonucleotide is as depicted in Figures and 1G and acts as both a primer and splice template. The second oligonucleotide is as depicted in Figure 1F.

Figure 1I depicts a target nucleic acid (RNA) which 35has a defined 5' terminus and has na additional sequences 5' to the target sequence, but has additional sequences to the target sequence.
The first oligonucleotide ~~2~9~$

comprises a primer without a promoter. The second oligonucleotide comprises an unblocked splice template which has a promoter 5' to its complexing sequence.
Figure iJ depicts a target nucleic acid (RNA) which has defined 5' and 3' terminus and no sequences besides.
the target sequence. The first aligonucleotide comprises a primer without a promoter. The second oligonucleotide is as depicted in Figure lI.
Figure iK depicts a target nucleic acid (RNA) which has a defined 5' terminus with no additional sequence 5' to the target sequence, but which has additional sequences 3' to the target sequence. The second oligonucleotide comprises a splice template having a promoter 5' to its complexing sequence, but which is blocked at its 3' terminus. The second oligonucleotide is incapable of acting as a primer.
Figure 1L depicts a target nucleic acid (RNA) which has defined 5' and 3' ends and no additional sequences besides the target sequence. The first oligonucleotide acts as both a primer and a splice template. The second oligonucleotide is a blocked splice template and is as depicted in Figure iK.
Figure 1M depicts a target nucleic acid (RNA) which has a defined 5'-terminus, and, thus, no additional sequences 5' to the target sequence, but which has additional sequences 3' to the target sequence. The first oligonucleotide is a primer as depicted in Figure 11. The second oligonucleotide is a blocked splice template having a promoter, as depicted in Figures iK and 1L.
Figure 1N depicts a target nucleic acid (RNA) which has both defined 5' and 3' sequences, having na additional sequences besides the target sequence. The first oligonucleotide comprises a primer without a promoter, as depicted in Figure iJ. The second oligonucleotide comprises a blocked splice template having a promoter, as depicted in Figures 1K, 1L and 1M.

~p~~95~

Figure 10 depicts a target nucleic acid (RNA) which has a defined 5' terminus, having no additional sequences 5' to the target sequence, but which has additional sequences 3' to the target sequence. One oligonucleotide is used for both the first and second oligonucleotide.
The oligonucleotide has a 3'-primer sequence which complexes to the 3'-terminal portion of the target sequence as shown in Step (1), and has a 5' splice template sequence with a promoter which complexes with the 3' terminus of the primer extension product as shown in step (4).
In summary, the methods of the present invention provide a method for autocatalytically synthesizing multiple copies of a target nucleic acid sequence without repetitive manipulation of reaction conditions such as temperature, ionic strength and pH which comprises (a) combining into a reaction mixture a target nucleic acid which comprises an RNA target sequence; two oligo-nucleotide primers, a first oligonucleotide having a complexing sequence sufficiently complementary to the 3' terminal portion of the RNA target sequence (for example the (+) strand) to complex therewith and a second oligonucleotide having a complexing sequence sufficiently complementary to the 3' terminal portion of the target sequence of its complement (for example, the (-) strand) to camplex therewith, wherein the first oligonucleotide comprises a first primer which optionally has a sequence 5' to the complexing sequence which includes a promoter and the second oligonucleotide co?nprises a primer or a splice template; provided that if the first oligonucleotide does not have a promoter, then the second oligonucleotide is a splice template which has a sequence 5' to the priming sequence which includes a promoter for an RNA polymerise; a DNA polymerise; an enzyme activity which selectively degrades the RNA strand of an RNA-DNA
complex (such as an RNAse H) and an RNA polymerise which recognizes the promoter. The components of the reaction mixture may be combined stepwise or at once. The reaction mixture is incubated under conditions whereby an oligonucleotide/target sequence is formed, including DNA
priming and nucleic acid synthesizing conditions (including ribonucleotide triphosphates and deoxyribo-nucleotide triphosphates) for a period of time sufficient whereby multiple copies of the target seguence are produced. The reaction advantageously takes place under conditions suitable for maintaining the stability of reaction components such as the component enzymes and without requiring modification or manipulation of reaction conditions during the course of the amplification reaction. Accordingly, the reaction may take plane under conditions that are substantially isothermal and include substantially constant ionic strength and pH.
The present reaction does not require a denaturation step to separate the RNA-DNA complex produced by the first DNA extension reaction. Such steps require manipulation of reaction conditions such as by substantially increasing the temperature of the reaction mixture (generally from.
ambient temperature to about 80°C to about 105°C), reducing its ionic strength (generally by 10X or moxe) or changing pH (usually increasing pH to 10 or more). Such manipulations of the reaction conditions often deleteri-ously affect enzyme activities, requiring addition of additional enzyme and also necessitate further manipulations of the reaction mixture to return it to conditions suitable for further nucleic acid synthesis.
Suitable DNA polymerases include reverse transcriptases. Particularly suitable DNA polymerases include AMV reverse ~Lranscriptase and MMLV reverse transcriptase.
Promoters or promoter sequences suitable for incorporation in the primers and/or splice templates used in the methods of the present invention are nucleic acid sequences (either naturally occurring, produced synthetically or a product of a restriction digest) that are specifically recognized by an RNA polymerase that recognizes and binds to that sequence and initiates the process of transcription whereby RNA transcripts are produced. The sequence may optionally include nucleotide bases extending beyond the actual recognition site for the RNA polymerase which may impart added stability or susceptibility to degradation processes or increased transcription efficiency. Promoter sequences for which there is a known and available polymerase that is capable of recognizing the initiation sequence are particularly suitable to be employed. Typical, known and useful promoters include those which are recognized by certain bacteriophage polymerases such as those from bacteriophage T3, T7 or SP6, or a promoter from E. coli.
Although some of the reverse transcriptases suitable for use in the methods of the present invention have an RNAse H activity, such as AMV reverse transcriptase, it may be preferred to add exogenous RNAse H, such as _E. coli RNAse H. Although, as the examples show, the addition of exogenous RNAse H is not required, under certain conditions, the RNAse H activity present in AM'V reverse transcriptase may be inhibited by components present in the reaction mixture. In such situations, addition of exogenous RNAse H may be desirable. 'Where relatively large amounts of heterologous DNA are present in the reaction mixture, the native RNAse H activity of the AMV
reverse transcriptase may be somewhat inhibited (see era., Example 8) and thus the number of copies of the. target sequence produced accordingly reduced. In situations where the target sequence comprises only a small portion of DNA present (era., where the sample contains signifi-cant amounts of heterologous DNA), it is particularly preferred to add exogenous RNAse H. fine such preferred RNAse H is E. coli RNAse H. Addition of such exogenous RNAse H has been shown to overcome inhibition caused by large amounts of DNA. (See, e.g., Example 8).

The RNA transcripts produced by these methods may serve as templates to produce additional copies of the target sequence through the above-described mechanisms.
The system is autocatalytic and amplification by the methods of the present invention occurs autocatalytically without the need for repeatedly modifying or changing reaction conditions such as temperature, pH, ionic strength or the Like. This method does not require an expensive thermal cycling apparatus, nor does it require several additions of enzymes or other reagents during the course of an amplification reaction.
The methods of the present invention may be used as a component of assays to detect and/or quantitate specific nucleic acid target sequences in clinical, environmental, forensic, and similar samples or to produce large numbers of copies of DNA and/or RNA of specific target sequence for a variety of uses.
In a typical assay, a sample to be amplified is mixed with a buffer concentrate containing the buffer, salts, magnesium, triphosphates, primers and/or splice templates, dithiothreitol, and spermidine. The reaction may optionally be incubated near 100°C for two minutes to denature any secondary structures in the nucleic acid.
After cooling, if the target is a DNA target without a defined 3' terminus, reverse transcriptase is added and the reaction mixture is incubated for 12 minutes at about 42°C. The reaction is again denatured near 100°C, this time to separate the primer extension product from the DNA
template. After cooling, reverse transcriptase, RNA
polymerase, and RNAse H. are added and the reaction is incubated for two to four hours at 37°C. The reaction can then be assayed by denaturing the product, adding a probe solution, incubating 20 minutes at 60°C, adding a solution to selectively hydrolyzes the label of the unhybridized probe, incubating the reaction six minutes at 60°C, and measuring the remaininc3 chemiluminescent label in a luminometer.

Several other methods for product determination may be employed in place of the in-solution probe hybridization.
If the target has a defined 3' terminus and one of the oligonucleotides i.s a splice template which has a complexing sequence sufficiently complementary to the 3' terminus to complex thearewith and a promoter sequence 5' to the complex sequence or the target is RNA, a typical assay includes mixing the target with the buffer concentrate mentioned above and denaturing any secondary structure. After cooling, reverse transcriptase, RNA
polymerase, and if deaired, RNAse H are added and the mixture is incubated for two to four hours at 37°C. The reaction can then be a:~sayed as described above.
II. Preliminary Procedures The following are several embodiments of preliminary procedures which optionally may be employed in conjunction with the preferred method of the present invention. Since some target nucleic acids require modification prior to autocatalytic amplification, these procedures may be employed to accomplish the modifications. Where the target nucleic acid (and target sequence) is originally DNA, these procedures may be employed to produce RNA
copies of the target sequence for use in the General Method. It should be appreciated that these Preliminary Procedures may themselves be repeated and therefore may be used as amplification methods in their own right.
3.0 Preliminary Procedure This method gives RNA copies of a target sequence of a target nucleic acid which comprises a (single-stranded) DNA with a defined 3' terminus. Preliminary Procedure I
uses two nucleic acid components: a target nucleic acid molecule and an oligonucleotide splice template. This procedure requires a DNA target nucleic acid having a deffined 3'-end. If they native 3' terminus is not known or 2(~2~9~~

is unsatisfactory for any reason, a new defined 3' terminus may be created by use of a restriction nuclease, ligation to another sequence, or some other means.
In the following description, (see Figs. 2A to 2C) the target nucleic acid will arbitrarily have the "minus"
sense. Thus, the splice template will have the "plus"
sense so as to be sufficiently complementary to the target to complex therewith. The splice template has a complexing sequence sufficiently complementary to the 3' terminus of the target to complex therewith. The splice template also has a sequence 5 ° to the complexing sequence which includes a promoter sequence for an RNA polymerise.
The splice template may optionally have other sequences 5' to the promoter, between the promoter arid complexing sequences, and/or 3' to the complexing sequence. The splice template may also be modified at the 3' terminus to be "blocked" so that it cannot be extended by adding additional nucleotides in an extension reaction and is rendered incapable of acting as a primer in addition to acting as a splice template.
Preliminary Procedure I uses two enzyme activities:
a DNA-dependent DNA polymerise and a DNA-dependent RNA
polymerise.
The target nucleic acid i.s treated with the splice template under conditions wherein an oligonucleotide/target sequence complex is formed and DNA
synthesis may be initiated. In a DNA extension reaction using an appropriate DNA polymerise, a sequence complementary to the sequence of the splice template 5' to the complexing sequence is added to the 3' terminus of the target DNA. The splice template, if not blocked at the 3' terminus, may also serve as a primer for the DNA polymer-ise and be extended to give a primer extension product.
The product of the extension reaction, either double-stranded or partially double-stranded, target/splice template complex acts as a template for the synthesis RNA
transcripts using an RNA polymerise which recognizes the 3 ~r promoter. The RNA transcripts may be then used for the general method or be used to generate DNA copies of the RNA as follows:
An RNA transcript comprising the target sequence (having the "plus" sense) is treated with a primer (which nominally has the "minus" sense) which has a complexing sequence sufficiently complementary to the 3' end of the target sequence of the RNA transcript to complex therewith under conditions whereby an oligonucleotide/target sequence complex is formed and DNA synthesis may be initiated. The primer is then extended in a DNA extension reaction using the RNA transcript as template to generate a DNA primer extension product having the target sequence.
The DNA target sequence is separated from the RNA
transcript by either denaturation or by degradation of the RNA and beginning with the splice template, the cycle is repeated. Optionally, the primer may also have additional sequences 5' to the priming sequence. The splice template may also have additional sequences 3' to the complexing sequence.
In one embodiment, the above method may be practiced using one oligonucleotide by using an oligonucleotide having a sequence which would comprise the primer 3' to the sequence which would comprise the splice template.
(See, eTa. Fig. 2C).
Preliminary Procedure I is further described by reference to Figures 2A to 2E. Figure 2A depicts a target nucleic acid (DNA) which has a defined 3' terminus, having no additional sequences 3' to the target sequence. The first oligonucleotide comprises bath a primer and a splice template and has a promoter 5' to its complexing sequence.
Figure 2B depicts a target nucleic acid (DNA) as shown in Figure 2A. The first oligonucleotide comprises a splice template which is blocked at its 3' end and is thus incapable of acting as a primer.
Figure 2C depicts a target DNA as shown in Figures 2A
and 2B. Figure 2C depicts the use of one oligonucleotide which has a splice template (with a promoter) sequence 5' to a primer sequence at its 3' end. Thus, the oligonucleotide acts as a blocked splice template in steps (l) and (7) and as a primer (and splice template) in step (4) .
Figure 2D depicts a target nucleic acid (DNA) which has a defined 5'-end and additional sequences 3' to the target sequence which undergoes prefatory complexing, primer extending and separating steps (steps 1 and 2) to generate a complementary DNA target having a defined 3'-terminus. The oligonucleotide of steps (1) and (7) comprises a primer which complexes with the 3' terminal portion of the target sequence of the original target DNA
(here nominally (+)). The other oligonucleotide (of steps (4) and (10)) comprises an unblocked splice template which complexes with the 3'-end of the complement of the original target.
Figure 2E depicts a target nucleic acid (DNA) which has a defined 5' terminus and additional sequences 3' to the target sequence which undergoes.prefatory complexing, extending and separating steps (steps (1) and (2)) to generate a complementary DNA target having a defined 3'-terminus. The oligonucleotide of steps (l) and (7) comprises a primer which complexes with the 3' terminal portion of the target sequence of the original target DNA
(here nominally (+)). The oligonucleotide of steps (4) and (10) comprises a blocked splice template which complexes With thed 3' terminal portion of the target sequence of the complement of the original target.
The splice template is complexed with the 3' terminus of the target nucleic acid under complexing conditions so that a target/splice template complex is formed and DNA
synthesis may be initiated. (step 1) A suitable DNA
polymerase is used to extend the 3' terminus of the target nucleic acid to add a sequence complementary to the sequence of the splice template 5' to the complexing sequence. Tf the 3' texminus of the splice template has 20~4~5~
3f not been blocked, and is sufficiently complementary to the target nucleic acid the splice template may act as a primer so that the 3° terminus of the splice template may also be extended. (Figure 2A) The resulting target/splice template complex may be either partially or completely double-stranded. (see Figure 2A versus Figure 2B and 2C) At minimum the double-stranded region comprises the promoter sequence and the priming sequence which complexed with the target nucleic acid.
The template of Step 2 is transcribed by an appropriate RNA polymerise. The RNA polymerise produces about 5-1000 copies of RNA for each template. Tn Figures 2A to 2C, the RNA produced is nominally of the °'plus"
sense and includes the sequence from the 3' end. of the promoter to the 5° end of the target nucleic acid.
The RNA product of Step 3 may be used according to the general method to autocatalytically amplify the target sequence or alternatively may be treated under complexing and priming conditions with a primer which has a complexing sequence at its 3' terminus sufficiently complementary to the 3' terminal portion of the target sequence in the RNA to complex therewith and optionally includes other sequences 5° to the complexing sequence.
The primer is extended using the RNA as a template by an RNA-dependent DNA polymerise in a primer extension reaction. This produces a product which is at least partially double-stranded and which must be made at least partially single-stranded for further synthesis by degradation of the RNA portion, such as by an RNAse H
(step 5) or by some other method such as denaturation.
The DNA produced in Step 6 may be used as a target molecule for Step 1 and treated with a splice template as described above to produce more RNA and. DNA. These steps may be cycled to produce any desired level of amplification. Tt should also be noted that, by appropriate choice of the splice template and primer(s), this new target molecule (Step s) may have different 20~0~~~
3~
termini than the original molecule. Furthermore, if the primer extension product from the RNA has a promoter sequence 5' to the complexing sequence, a second primer having a complexing sequence of the "plus" sense and optionally other sequences 5' to the complexing sequence may be used to copy the extended primer product to produce a double-stranded template for an RNA polymerase. A
template so produced enables the RNA polymerase to make "minus" sense RNA which may be amplified using the general method or further amplified using the procedures herein.
Preliminary Procedure II
Preliminary Procedure II differs from Preliminary Procedure I in the way the autocatalytic species is gener ated. The DNA target nucleic acid need not have a defined 3' terminus. In one aspect, a primer containing a promoter sequence 5° to the complexing sequence is used instead of a splice template to introduce the promoter sequence into the template for the RNA polymerase. The primer has a complexing sequence sufficiently complementary to the 3'-terminal portion of the target sequence to complex therewith and a sequence which includes a promoter for an RNA polymerase 5' to the complexing sequence. The primer is extended with a DNA
polymerase to give a primer extension product.
After separation of the strands, usually by thermal denaturation, a second oligonucleotide of the same sense as the target molecule is used as a primer to synthesize a DNA complement to the first primer extension product.
The second primer may have additional sequences 5' to the complexing region which may include a promoter. Inclusion of a promoter sequence in the second primer may enhance amplification. The second primer may also be a splice template.
Preliminary Procedure II is further described with reference to Figure 3. Figure 3 depicts a target DNA
which has additional sequences both 5' and 3' to the target sequence. The first primer has a complexing sequence at its 3' terminus and a sequence 5' to the complexing sequence which includes a promoter sequence and complexes sufficiently with the target to serve a priming function and initiate DNA synthesis, An appropriate DNA
polymerase extends the primer to give a primer extension product. The strands are separated by denaturation or other means to yield to a single-stranded promoter containing primer extension product.
A second primer is used which has a complexing sequence at its 3° terminus sufficiently complementary to the 3' terminal portion of the target sequence of the first primer extension product and, optionally, other sequences 5' to the complexing sequence which may include a promoter sequence. The second primer is complexed sufficiently with the primer extension product from Step 3 to serve a priming function and initiate DNA synthesis.
The DNA polymerase extends the second primer to give a second primer extension product. The resulting double-stranded DNA molecule may now serve as a template for the RNA polymerase to generate RNA transcripts for the General Method. As depicted in Figure 3, the RNA molecules produced are nominally of the °°plus" sense and may be multiplied using the general. method of the present invention.
Where the complexing sequence of the second primer is complementary to the 3' terminus of the first primer extension product from Step 3 and the secand primer includes a promoter sequence 5° to the complexing sequence, the second primer may serve as a splice template so that the 3'-terminus of the first primer extension product from Step 3 may be further extended to add the promoter sequence and produce a template far the RNA
polymerase which produces RNA transcripts of bath senses.
The RNA molecules so produced may be amplified using the general method.

'~Q~~9~~

In another aspect of the present invention, the second primer acts as a splice template and has a promoter 5' to the complexing sequence, so that the first primer need not nave a promoter. In that case, the first primer extensian product from Step 2 is further extended to produce a complement to the promoter sequence, thus generating a template for the production of "minus" sense RNA by the RNA polymerase.
By repeating the steps described above, additional RNA and DNA copies of the target sequence may be produced.
Examples Preface The following examples of the pracedures previously described demonstrate the mechanism and utility of the methods of the present invention. They are not limiting to the inventions and should not be considered as such.
Many of the materials used in one or more examples axe similar. To simplify the descriptions in the.
examples, some of the materials will be abbreviated and described here.
The template referred to as "frag 1" is a double stranded segment of DNA homologous to a region of DNA from the hepatitis B genome. It has been excised from a plasmid via restriction nuclease digestion arid purified from the remaining nucleic acid by chromatographic methods. Subsequent to purification, the fragment has been cut with a restriction endonuclease and purified via phenol extraction and ethanol precipitation to yield the desired taxget.
The template referred to as "M13L(-)" is a purified single-stranded DNA target containing, as a small fraction of the total sequence, the minus strand target sequence.
Several different primers and splice templates were used in the examples described herein. The oligonucleotide referred to as T7pro (+~) contains, near the 2~~~~a8 5' terminus, a T7 RNA polymerase promoter and, near the 3' terminus, a sequence complementary to the 3' terminus of the minus-strand target sequence to the two templates described above; T7pro(+) also contains other sequence 5 information to enhance performance. The sequence for T7pro(+) is 5'-AATTT AATAC GACTC ACTAT AGGGA GAGGT TATCG
C*TGGA* TGTGT CTGCG GC*GT3'.
Another oligonucleatide similar to T7pro(+) is ddT7pro(-H) . It differs from T7pro(-~-) in that the 3' 10 terminus has been extended with a dideoxy nucleotide using terminal deoxynucleotidyl transferase. Unlike T7pro(+), ddT7pra(+) is incapable of serving as a primer for DNA
synthesis by reverse transcriptase but can act as a splice template.
15 HBV (-) Pr is a primer which will hybridize to the plus strand of the frag 1 template and is homologous to a sequence within the M13L(-). [HBV(-)Pr is complementary to a sequence 3° to the plus strand sequence homologous to the T7pro(+).] The sequence for HBV(-)Pr is 5'-GAGGA
20 CAAAC GGGCA ACATA CCTTG-3'.
Another oligonucleotide containing a promoter region is T7pro(-). This promoter-primer contains a sequence identical to T7pro(+) but replaces the sequence complementary to the minus target with a sequence 25 complementary to the plus target. The 3' terminus of T7pro(-) is complementary to the 3° terminus of the plus strand of frag 1. The sequence for T7pro(-) is 5°-AATTT
AATAC GACTC ACTAT AGGGA GATCC TGGAA TTAGA GGACA AACGG
Gc-3'. Like the ddT7pro(+), ddT~pro(-) is a 3' blocked 30 oligonucleotide made by extending the 3° terminus with a dideoxynucleotide using terminal deoxynucleotidyl transferase. The ddT7pro(-) cannot serve as a primer but is otherwise similar to T7pro(-).
The templates used in these examples contain sub 35 stantial sequence between the regions homologous or complementary to the primers and splice templates described above. As 'the sequence between the oligonucleotides will be amplified as a result of the invention, quantification of this sequence provides a specific means of measuring the amplification. It has been convenient to assay the products by hybridization techniques using DNA ;probes specific for the sequences coded for between the oligonucleotide primers and splice templates. Two probes are used in the examples presented below: Probe(+) and Probe(-). Probe(+) is complementary to the minus sense product and Probe(-) is complementary 1.0 to the plus sense product. The sequence for Probe(+) is 5'-CCTCT TCATC CTGCT GCTAT GCCTC-3' and the sequence for Probe(-) is 5'-GAGC A,TAGC AGCAG GATGA AGAGG-3'. The probes used herein have been labeled with a chemiluminescent tag. In the assay, the label on hybridized probe emits light Which is measured in a luminometer.
In the following e:Kamples, relative amplification was measured as follows. A sample of the amplification reaction mixture (usually 10 ~cl) was diluted to 50 ~1 with lOmM Tris-HC1, pH 8.3, and denatured two minutes at 95°C.
After cooling on ice, 5~0 ~1 of a probe solution captaining approximately 75 fmol Probe(+) or Probe(-), 0.2 M lithium succinate, pH 5.2, 21% (w/v) lithium lauryl sulfate, 2 mM
EDTA, and 2 mM EGTA, was added to the sample and mixed.
The reactions were then incubated 20 minutes at 60°C and cooled. To each hybridization reaction was added 500 ~1 of a solution prepared, by adjusting a saturated sodium borate solution to pH 8.5 with hydrochloric acid, then diluting to bring the borate concentration to 0.8 M final and adding Triton X-100 to 5% (v/v) final. The reactions were then mixed and incubated six minutes at 60°C to destroy the chemiluminescent label of the unhybridized probe. This method of destruction of the chemilumi-nescent label of unhyb~ridized probe is quite specific;
3~~ only a very small fraction of the unhybridized probe remains chemiluminescent. The reactions were cooled and the remaining chemilu:minescence was quantified in a * Trade-mark luminometer upon the addition of 200 ~1 of 1.5 M sodium hydroxide, 0.1% (v/v) hydrogen peroxide. In the assay, hybridized probe emits light which is measured in a luminometer. Since the reaction which destroys the chemiluminescent label of unhybridized probe is not 100%
effective, there is generally a background level of signal present in the range of about 300 to 1300 relative light units (RLU).
Many other assay methods are also applicable, including assays employing hybridization to isotopically labeled probes, blotting techniques and electrophoresis.
The enzymes used in the following examples are avian myeloblastosis virus reverse transcriptase from Seikagaku America, Inc., T7 RNA polymerase from New England Biolabs or Epicentre, and Moloney murine leukemia virus (MMLV) reverse transcriptase and _E. coli RNAse H from Bethesda Research Laboratories. Other enzymes containing similar activities and enzymes from other sources may be used; and other RNA polymerases with different promoter specificities may also be suitable for use.
Unless otherwise specified the reaction conditions used in the following examples were 40 mM Tris-HCl, 25 mM
NaCl, 8 mM MgCl~, 5 mM dithiothreitol, 2 mM spermidine trihydrochloride, 1 mM rATP, 1 mM rCTP, 1 mM rGTP, 1 mM
rUTP, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, 0.15 WM each primer or splice template, and specified amounts of template and enzymes in 100 ~,l volumes.
These reaction conditions are not necessarily optimized, and have been changed as noted for some systems. The oligonucleotide sequences used are exemplary and are not meant to be limiting as other sequences have been employed for these and other target sequences.

Example 1 Preliminary Procedure I
To demonstrate that this system worked, each of the four promoter containing oligonucleotides described above were each respectively put into reactions with or without 4 fmol target (frag 1). The reaction was incubated 2 minutes at 95°C to denature the target, then cooled to allow the oligonucleotides to anneal to the target.
Reverse transcriptase, 13 Units, and T7 RNA polymerase, 100 Units, were added and the reaction was incubated 30 minutes at 37°C. One-tenth of 'the reaction was assayed using the hybridization method. The results (in Relative Light Units or ''RLU(s)" and fmols) presented in Table 1 show that both the blocked and unblocked oligonucleotides serve as splice templates to produce product. The signals measured for reactions without target represent typical background levels of signal, for this type of assay.
Table 1. Comparison of Splice Templates in Preliminary Procedure I.
Product Spice Template Target Probe(,-) RLU fmol ddT7pro(+) 4 fmol Probe(-) 38451 240 ddT7pro(+) 0 fm01 Probe(-) 544 0 T7pro(+) 4 fmol Probe(-) 65111 410 T7pro(+) 0 fmol Probe(-) 517 0 ddT7pro(-) 4 fmol Probe(+) 47756 85 ddT7pro(-) 0 fmol Probe(+) 879 0 T7pro(-) 4 fmol Probe(+) 156794 290 T7pro(-) 0 fmol Probe(+) 600 0 Example 2 Cycling with Preliminary Procedure I
The amplification system was cycled with ddT7pro(+) and T7pro(+). In this experiment, 4 amol frag I, HBV(-)Pr and ddT7pro(+) or T7pro(+) were mixed in standard re-actions and incubated at 95°C. After cooling, 13 Units of reverse transcriptase and 100 units of T7 RNA poly-merase were added and the mixture was incubated 30 minutes at 37°C. One-tenth of the reaction was removed for assay and the cycle was repeated with the remainder. After repeating the cycle a third time, the 10,1 aliquots were assayed by the hybridization method using Probe(-). The results presented in Table 2 indicate that product is amplified through cycling with both blocked and unblocked splice templates.
Table 2. Cycling with Preliminary Pracedure I.
Splice templateTarget Relative LightUnits(RLU's) Cycle Cycle2 Cycle 3 ddT7pro(+) amol 602 1986 10150 ddT7pro(+) amol 658 592 595 T7pro(+) 4 amol 891 6180 52160 T7pro(-t-) amol 496 504 776 Example 3 Sensitivity of Preliminary Procedure I
In this example the unblocked splice template, T7pro(+), and the primer, HBV(-)Pr, were used to test the sensitivity of the amplification method. Six cycles of Preliminary Procedure I were run as described in Example 2 with decreasing quantities of frag 1. After amplifi-cation, the product was assayed using the hybridization method described in the Detailed Description of the Invention. Using this method, 4x102 moles frag 1 could be detected (see Table 3).

~o~o~~~
Table 3. Sensitivity using 6 cycles of Preliminary Procedure T.

Target Sample Product (moles) ~.l RLU's 5 4x10 ~8 5 328390 4x10 ~9 20 105364 4x10 Z~ 20 3166 4x10 z~ 20 1927 Example 4 Amplification Including Prelimina~r Procedure I
In the following example, the target to be amplified was frag 1. In the first set of reactions, various combinations of target and.splice templates were incubated at 95°C for two minutes then cooled prior to adding 13 Units of reverse transcriptase and 100 Units of T7 RNA
polymerase. The reactions were incubated 30 minutes at 37°C then 5~1 aliquots of the reactions were assayed with both probes to quantitate the products. Subsequent to this assay, reactions were prepared using 5~e1 of reactions 1 and 2 and the T7pro(-) splice template. The mixtures were incubated 2 minutes at 95°C then cooled prior to adding 13 Units of reverse and 100 Units of T7 RNA
polymerase. The new reactions were then mixed and incu-bated at 37°C for 2 hours. Aliquots of 101 were removed to an ice bath at time points indicated in Table 4 below.
The products were assayed using the hybridization method previously described. The data indicate that both splice templates allow production of RNA from frag 1. The data also indicate significantly more minus and plus sense products are produced in the reactions containing RNA and the splice template complementary to that RNA. And, finally, the reaction kinetics for the reaction 1B show a geometric increase in product whereas the kinetics for the 2U reaction are of a more linear form. This difference indicates the product in the 1B reaction is serving as a substrate to generate more products thus the reaction is autocatalytic.
Table 4. Preliminary Procedure I
Reaction 1 2 3 4 Target fmol) Yes Yes Yes No (4 T7pro(+) Yes No No No T7pro(-) No No Yes No Probe Time Relative LightUnits(RLU's) Probe(+) 30' 1156 1058 21859 591 Probe(-) 30' 11693 771 744 691 Reaction 1A 1B 2A 2B

Reactionl Yes Yes No No Reac~tion2 No No Yes Yes T7pro(-) No Yes No Yes Probe Time Relative Light Units Probe(+) 0' 714 757 639 661 30' 686 339 1663 1373 60' 718 6331 645 1786 120' 816 16889660 2238 Probe(-) 120' 3142 6886 637 780 Exam~~le 5 Reaction Kinetics for Amplification Including Preliminary Procedure I
The following example further demonstrates the poten tial of this embodiment of the methods the invention. A
small quantity of frag 1 (10 amol), was used in reactions with various combinations of T7pro(+), T7pro(-), and HBV(-)Pr. The reaction mixtures were incubated at 95°C
for two minutes to denature the DNA target and cooled prior to adding reverse transcriptase and T7 RNA polymer-ase. After mixing, the reactions were incubated 30 minutes at 37°C. Fifteen microliter aliquots were removed at various time points and stored at 0°C until assayed.

The hybridization assay was used to quantitate the products of the reactions. The data presented in Table 5 show that the invention requires one splice template and one primer. A second splice template is advantageous, however. The results with only one primer or splice template were below the detection limits of the assay. As in the previous example, the reaction kinetics are geometric, indicating an autocatalytic system.
Table 5. Preliminary Procedure II Reaction Kinetics.
Reaction 1 2 3 4 5 6 7 Target amol)No Yes Yes Yes Yes Yes Yes (10 T7pro(+) Yes No Yes No No Yes Yes T7pro(-) Yes No No Yes No No Yes HBV(-)Pr Yes No No No Yes Yes No Time (minutes) Minus (RLU's) Product 180 678 635 714 930 62'72171978682 Time (minutes) Plus Product (RLU's) Our subsequent work has demonstrated continued product synthesis for over 5.O hours, which is substantially better than prior art methods. We also have demonstrated increased sensitivity.

2~~~~~~

Exa_~ple 6 Am~lifiaation Tncluding Preliminary Procedure II
In this example various combinations of primers were used to amplify 500 amol of a DNA target without defined termini. The target was the M13L(-) referenced above.
Upon reaction preparation, the samples were incubated two minutes at 95°C, then cooled prior to adding 13 Units of reverse transcriptase. The reactions were then incubated twelve minutes at 42°C. Next the reactions were again heated for two minutes at 95°C and cooled. Reverse tran-scriptase and T7 RNA polymerase were added and the reactions were incubated fox two hours at 37°C. Ten microliter aliquots of the reaction were assayed with both Probe(+) and Probe(-). The results presented in Table 6 show that synthesis of large amounts of nucleic acid occurs only when two primers are employed. They also demonstrate the benefit of two promoter-primers over one promoter-primer and one primer. The low level synthesis in reactions 4 and 5 correspond to synthesis of approxi-mately one copy of DNA from the original template. The system employs an initial 95°C denaturation step which may serve to denature double-stranded targets or double-stranded regions of a single-stranded target as well as inactivate unwanted nuclease and protease activities.
Table 6. Preliminary Procedure II System.
Reaction 1 2 3 4 5 6 7 M13L(°) No No Yes Yes Yes Yes Yes T7pro(+) Yes Yes No Yes No Yes Yes T7pro(-) Yes No No No Yes Yes No HBV(-)Pr No Yes No No No No Yes Probe Relative Light Units (RLU's) Probe(+) 862 744 762 1089 2577 96221 30501 Probe(-) 473 420 483 3038 1080 15171 14863 Example 7 Effect of RNAse H
To demonstrate that the addition o.f exogenous RNAse H may improve amplification in the autocatalytic systems described of the present invention, several reactions were prepared using various quantities of target, M13L(-), and either 0 or 2 Units of exogenous RNAse H. The exogenous RNAse H used was derived from E, coli. All reactions were 1o prepared with T7pro(+) and T7pro(-). The reactions were subjected to the 95°C denaturation and cooled prior to adding reverse transcriptase. After a twelve minute incubation at 42°C, the reactions were again denatured at 95°C. After cooling, reverse transcriptase, T7 RNA
polymerase, and, if indicated (see Tables 7), RNAse H was added and the reactions were incubated for 3 hours at 37°C. Aliquots of 10,1 were removed from each reaction at hourly intervals for assay by the hybridization method.
The data in Table 7 snow that exogenous RNAse H signi-ficantly enhanced the reaction kinetics and increases the.
sensitivity of the invention. Signals less than 600 RLUs were interpreted as typical background levels.
Table 7. Effect of Exogenous RNAse H on Amplification Including Preliminary Procedure II Sensitivity.
Target RNAse H RLU's Time(hours) at (moles) (Units) 0 1 2 3 _ 1x10 0 478 7659 28716 60443 ~~

_ 0 440 946 13332 36039 1x10 _ 0 413 581 10063 41415 1x10 _ 0 406 424 717 4520 1x10 2~

1x10 2 419 20711 50389 64073 _ 2 411 6831 21312 29818 1x10 _ 2 420 604 1281 1375 1x102 ~~2~~5~
Examgle 8 Effect of Exogenous DNA
It has been demonstrated that the addition of exogenous DNA may significantly inhibit this autocatalytic 5 amplification system. To further demonstrate the benefit of adding exogenous RNAse H, amplification reactions were prepared with or without 2 ~g calf thymus DNA to demonstrate this inhibition. In reactions with the calf thymus DNA, two concentrations of reverse transcriptase 10 were employed to test whether additional AMV RNAse H would overcome the inhibition. Also, RNAse H from E. coli was added to some of the reactions fox the same reasons. The reactions differ from the standard reactions in that the concentration for each of the ribonucleotides were 15 increased to 2.5 mM and the concentration of magnesium chloride was increased to 12.8 mM. The reactions were prepared using 100 amol of M13L(-) as a target and T7pro(+) and T7pro(-). After denaturing two minutes at 95°C, the reactions were cooled and 13 or 39 Units of 20 reverse transcriptase were added and the reactions were incubated 12 minutes at 37°C. The reactions were again denatured at 95°C and cooled prior to adding l3 or 39 Units of reverse transcriptase, 100 or 300 units of T7 RNA
polymerase, and either 0 or 2 Units of E. coli RNAse H.
25 After incubating one hour at 37°C, 10 ~1 of each reaction was assayed using the hybridization assay. The results presented in Table 8 showed that the calf thymus DNA
inhibited the reaction by 90% in comparison to a reaction system without exogenous DNA and that additional reverse 30 transcriptase (and its associated RNAse H) did not significantly affect the product amplification. The addi-tion of more T7 RNA polymerase did have a significant effect on the product yield, but it was small relative to the increase due to addition of exogenous RNAse H. Not 35 only was the inhibition eliminated, the amplification was increased aver five-fold relative to the reaction without the calf thymus DNA and E. coli RNAse H. The signals ~p~p958 observed with the higher amount of E. coli RNAse H were saturating for the amount of probe used in the hybridiza tion assay. To accurately quantitate these samples, dilu tion of the amplified product would be required before assaying.
Table 8. Effect H Amplification of RNAse on Inhibition by Exogenous DNA

Reverse T7 RNA E. coli Target Relative Exogenous Transcriptasepolymerise H DNA DNA Light RNAse (Units) (Units) (Units) (ug) Units (amol) 13 100 0 100 2 . 3003 Example 9 Amplification By the General Method This system does not require an initial transcription and denaturation; a DNA complement of the target is made and the original target is removed by the RNAse H. The DNA may then anneal to a second primer or promoter-primer and through DNA synthesis produce a template for the RNA
polymerise. Tf the RNAse H is not active, the DNA: RNA
hybrid produced first will end the reaction without producing a template for the RNA polymerise. Tn an attempt to demonstrate the method of this invention, a plasmid containing an RNA polymerise promoter and the target nucleic acid was used to produce large quantities of single-stranded RNA transcripts containing the target 2~~0~~~

sequence within other sequences. Two similar reactions were also prepared: one containing the plasmid without the RNA polymerise and 'the other with the RNA polymerise without the plasmid. Oilutions of each of these reac-Lions were assayed to quantitate the products. Equivalent dilutions of all three reactions were used to prepare amplification reactions containing the two promoter-primers, T7 pro(+) and T7 pro(-). Reaction 1 of Table 9 contained 60 amol of RNA and 0.6 amol of the starting plasmid. Reaction 2 captained 0.6 amol of the starting plasmid, but no RNA. Reaction 3 contained no target. The reactions were not denatured at 95°C; instead reverse transcriptase and T7 RNA palymerase were added and the reaction was incubated at 37°C for four hours. Aliquots were remaved hourly for later assay by the hybridization method. As shown in Table 9, the reaction containing the plasmid and the RNA produced from the plasmid gave large hybridization signals; the reaction containing the plasmid alone produced significant product as the T7 RNA polymer-ase could produce RNA from the plasmid which could then be utilized according to the General Method to produce more nucleic acid of both senses; and, finally, the control (Reaction 3) containing no target produced nothing.
Table 9. Preliminary Procedure IV Reaction Kinetics.
Time Reaction 1 2 3 1 2 3 (hours) RLU's for Probe(+) RLU's for Probe(-) Rxn 1: plasmid plus RNA.
Rxn 2: plasmid.
Rxn 3: no target Example 10 Amplification by the General Method The following experiment was done to determine if other methods of initiation were possible for the invention which may alleviate the need for the first primer extension and denaturation step for DNA targets without defined termini. In this experiment, the stated quantities of target are amplified With or without the first reverse transcriptase step as indicated in Table 10.
The amplification time once all enzymes are added was four hours. The target used was the M13(+) diluted into normal saline. T7pro(+) and T7pro(-) were used. To 25 ~,1 of each dilution was added 25 ~S1 O.1N KOH. The samples were incubated ten minutes at 98°C and then cooled. Each sample was brought to 100 dal and final concentrations of 50 mM Trizma* base, 40 mM glutamic acid, 25 mM potassium hydroxide, 12.8 mM magnesium chloride, 5 mM
dithiothreitol, 2 mM spermidine trihydochloride, 2.5 mM
each ribonucleotide tri.phosphate, 0.2 mM each deoxyribo-2~7 nucleotide triphosphate, and 0.15 uM each primer. To the Standard Protocol tubes, 13U reverse transcriptase was added and the reactions were incubated 12 minutes at 42°C, then two minutes at 95°C, and cooled. Then to all tubes was added 13U rever:~e transcriptase, 100U T7 RNA
2!5 polymerase, and 0.5U R~NAse H. The reactions were then incubated four hours at 37°C and 10 ~1 aliquots were assayed using the che:miluminescent probe assay. The results presented in Table 10 show that some amplification was evident using the abbreviated protocol. And although 30 the level of amplification observed was significantly less than that for the Standard Protocol, this may be further developed to be as efficient or may be useful in cases where significant levels of target nucleic acid are present.
3 °i * Trade-mark 20~~19~~

Table 10. Amp?ification Without First Primer Extension Target Moles Protocol RLU's M13(+) 3.8E-17 Standard 2295451 " 3. SE-19 " 2374443 " 3.8E-21 " 230227 Negative 0 " 3037 M13~+) 3.8E-17 Short 2475574 " 3.8E-19 " 27209 " 3.8E-21 " 17144 Negative 0 " 1679 The likely explanation for these data is that T7RNA
polymerase is not completely specific. fihere is a low level of random RNA synthesis which generates small numbers of RNA copies of the target regions. These copies are amplified via the standard method.
Our initial work demonstrated excellent amplification with certain preimer sets and targets without the addition of exogenous RNAse H. Our subsequent work clarifying the.
mechanism of the reaction has made it possible to efficiently apply the method to a wider variety of targets. We disclose and claim herein methods which both use exogenous RNAse H in amplification and those which rely on RNAse H activity associated with reverse transcriptase.
We have discovered that E. coli RNAse should not routinely be added as it does not always improve amplification. We have determined that some forms of RNAse H are sequence specific as to where they cut the RNA
of RNA:DNA hybrids. Tn the amplification reaction, we have not detected the promoter- containing primer in full-length product DNA. In embodiments using two promoter-primers, only one of the promoter primers is detestably incorporated into full-length product DNA. All other mechanisms that have been postulated by those skilled in the art show full-length product DNA containing both primers. We attribute these findings to the likelihood that RNAse H is not fully degrading the major RNA species synthesized during amplification. Based on these findings, a new amplification mechanism is set forth herein which incorporates our findings regarding RNAse H
sequence specificity. New and useful promoter-primer design criteria are disclosed. Furthermore, we claim herein novel methods fox synthesizing multiple copies of a target nucleic acid sequence, comprising 1) selecting a primer, complementary to a portion of the RNA target sequence, which complexes with the portion of the RNA target, said portion of the target located such that it remains capable of forming primer extension product after being exposed to degradation by a selected RNAse H;
2) selecting a promoter-primer complementary to a portion of the DNA to be obtained by extension of the primer, which complexes with the DNA in an area where substantially all of the complementary RNA is removed from the duplex through degradation of RNAse H; and 3) combining the RNA target with. the primer, promoter-primer, RNAse H, reverse. transcriptase and transcripase and forming multiple copies of the RNA and multiple copies of DNA complementary to the RNA. The novel methods herein described do not make a substantially equivalent number of copies of DNA of the same polarity (or "sense") as the RNA target sequence.
This procedure provides a method which permits the design of efficient promoter-primers and primers for new target sites. We disclose and claim herein such promoter primer and primer combinations and design criteria for same. The mechanism disclosed herein involves a novel reaction intermediate for transcription of RNA strands.
This intermediate is a double-stranded complex of RNA and DNA which contains a double-stranded DNA promoter region.
Nothing like this has, to our knowledge, ever been described in the literature. Tndeed, none of the prior ~~2~9~'~
5s art systems, specifically neither Guatelli, J.C. et al., 87 PNAS 18?4-1878 (1990), nor PCT App. Ser. No. 88/10315 to Gingeras, T.R. et al., properly select the primer and promoter-primer sequence based upon the location of RNAse H degradation sites. Thus, the methods herein axe novel and nonobvious from any previously disclosed.
Recognition of the importance of the RNAse H sequence specificity and an understanding of the reaction mechanism is key to the efficient application of this target amplification method to a wide variety of target sequences. Moreover, until this time, practitioners assumed that RNAse H fully and systematically degraded the RNA strand of an RNA: DNA complex.
T. The Mechanism of Amplification Methods Testing amplification efficiency with both prior art methods revealed a great deal of variability in amplification efficiency with small changes in the primer sets used. The reaction method generally accepted in the prior art did not, in our view, provide a reasonable explanation as to why one primer set worked so much better than another.
Attempts to improve amplification using the prior art methods did not give satisfactory results. Efficiency of priming was examined to see if differences in the ability to initiate DNA chains were responsible for observed differences in primer set efficiency. No correlation between priming efficiency and overall amplification could be found: Analysis of primer sets for the ability to form self-complementary structures and cross-complementary structures indicated that differences in primer efficiency were not solely attributable to these factors either.
We also found that the addition of E. coli RNAse H
did not uniformly improve amplification. As the data submitted herein show, the results observed varied from target to target and from primer set to primer set. The amount of E. coli RNAse H added also is very important and 2 fl~095$

must be kept within a narrowly defined range. With a given target and primer set, addition of _E. coli RNAse H
is helpful in some cases when the reverse transcriptase is that from avian myeloblastosis virus ("AMV") but not when the reverse transcriptase is that from Moloney marine leukemia virus ('°MMLV"). Data illustrating these conclusions are provided herein.
Earlier work suggested that AMV reverse transcriptase leaves relatively large fragments when it digests the RNA
from the RNA: DNA hybrid formed during virus replication.
These fragments serve as primers to initiate synthesis of the second DNA strand. We report herein our findings that there is evidence for sequence specificity of the AMV and MMLV RNAse H activities.
In order to elucidate the mechanism of the reaction, individual primers were terminally labeled with 3zP, and the incorporation of each primer into DNA products was examined by polyacrylamide gel electrophoresis. According to the generally accepted prior art mechanism, both primers would be incorporated into full length DNA
products. Our experiments showed, however, that only the primer complementary to the major RNA species synthesized during amplification was incorporated into full length product. The primer having the same polarity as the major RNA strand was never detected in full length DNA product.
In fact, it remained quite small. These results were confirmed with a number of different targets and primer sets.
The failure to detect extension of one of the primers indicated that a fully double-stranded DNA intermediate did not accumulate during amplification, and was not required fox autocatalytic amplification. These observations indicate a mechanism for the amplification systems of this invention which takes into account probable sequence specificities of the RNAse H. The mechanism is generally depicted in Fig. 4.

Experiments have shown that the enzyme cuts the RNA
of an RNA: DNA hybrid into specific pieces. Furthermore, the locations of the cut sites were shown to be in specific regions. To confirm the mechanism, an RNA: DNA
hybrid was prepared which contained the plus strand RNA
that would be generated from our T7pro''/T7pro- target and primer set combination. The 3zP labelled RNA was hybridized to complementary DNA, incubated with AMV
reverse transcriptase (containing its associated RNAse H
activity), and the reaction products were analyzed by polyacrylamide gel electrophoresis (Figure 5). The fragment size indicated that several small pieces were generated and that these were produced by cuts near one or both ends. The interior region of the molecule was not cut. This experiment demonstrates that the enzyme has sequence or structural specificity under the reaction conditions used. The results were entirely consistent with the reaction mechanism of Fig. 4.
Further experiments were performed to determine where z0 the cut sites occurred. It is preferred that multiple cut sites occur in the region homologous to the promoter containing primer and not in the region binding the other primer. By labeling the termini of the RNA individually and analyzing the digestion products, it was found that under the conditions used the cuts were detected only at the 5' end of the RNA. This is consistent with the mechanism of Fig. 4.
Sequencing experiments were performed to determine the sequences at which the RNAse H activities of AMV and i~ILV reverse transcriptases cut. Sequences were identified that were specifically cut by each enzyme. As predicted, the sequence specificities of the two enzymes are different, and this is believed to explain why some primer sets work better with one enzyme and some with another. The sequence specificities obtained for the MMLV
enzyme under our reaction conditions do not match those reported in the literature under another set of reaction 2~~~~~~

conditions, indicating that specificity may be influenced by reaction conditions.
Scrutiny of the role of the RNAse H in the amplifica tion mechanism has resulted in our finding that completely removing the promoter directed transcript from its cDNA
copy may not be necessary, or even desirable for formation of a new transcriptionally active template. Thus, in some applications, even a very low level of RNAse H activity, deriving from the reverse transcriptase-intrinsic RNAse H, will be sufficient for effective amplification if the RNase H is more site selective in allowing the promoter-primer to anneal to the first strand cDNA product or if it interferes less with the annealing of the other primer to the transcript.
Since E. coli RNAse H is reportedly less specific than the retroviral enzymes, it may cleave in the region to which the non-promoter containing primer binds, especially if the concentrations of this primer, the target, the E. coli RNAse H, and components affecting the enzyme activity are not. carefully balanced. In our view these results make the use of E-coli RNase H non-preferable in commercial applications. Addition of another RNAse H activity, one with different specificities, may be useful in those cases in which the reverse transcriptase RNAse H does not cut in the desired regions or does not cut under appropriate conditions.
Work with MMLV reverse transcriptase, for example, has shown that this enzyme is less sensitive than the AMV
enzyme to inhibition by sample DNA. It is the best mode for many systems.
New primer sets were designed and are set forth herein based upon the model and the RNAse H sequence specificity information that we have obtained to date.
Significantly better synthesis was obtained from these primex sets than was obtained with those designed previously without knowledge of the mechanism and sequence specificities. The invention herein described makes 202Q95~
possible the design of functional primer sets for specific target regions.
The new mechanism we have discovered involves a novel reaction intermediate for transcription of RNA strands.
5 This intermediate is a double-stranded complex of RNA and DNA which also contains a double stranded DNA promoter region. To our knowledge, the reaction is demonstrably different from any previously disclosed.
An understanding of the reaction mechanism is 10 critical to using these target amplification procedures.
Recognition of the importance of the RNAse H sequence specificity is key to the efficient application of this target amplification method to a wide variety of target sequences. On the other hand, the empirical approach to 15 promoter-primer design is very intensive, costly, and has a law frequency of success, making this invention a useful advance in the art.
A. Narrow Ranae of Activity for RNAse H Concentration 20 The amplification system with E. coli RNAse H
initially was considered to be the preferred embodiment because greater synthesis was achieved with the particular target and primer set being studied, and the E. coli RNAse H was found to be useful in helping to overcome inhibition 25 by sample DNA. However, analysis of the reaction indicates that addition of E. call RNAse H is detrimental to amplification in many cases. Moreover, amounts of E.
col' RNAse H added must be carefully controlled aver a narrow range since the presence of too much or too little 30 is harmful. For practical commercial application of the method, this is a significant drawback, especially since the enzyme may not be completely stable on storage. In addition, the use of E. coli RNAse H adds significant cost and complexity to the system. The cost may be prohibitive 35 for many commercial applications. TJsing E. coli RNAse H
makes the assay more complex, which in turn increases research and development costs and may make the assay too ~~~~9~~

delicate for wide commercial application. Thus, our elucidation of the assay mechanism has resulted in methods for a widely applicable assay both in terms of technical feasibility (applicability to target sites) as well as being a cheaper and a more robust procedure. Since the addition of E. coli RNAse H was found to result in increased amplification in early experiments, the effect of E, cot RNAse H on the performance c~f the amplification system in samples containing serum or human DNA was examined in several experiments.
Example 11 Optimization of E. coli RNAse H Concentration Experiments were performed to determine the amount of E. coli RNAseH needed for optimal amplification in serum.
The following experiment compared amplification with the T7pro+/T7pro~ primer pair in the presence of 0, 0.25, 0.5 and 1 U of RNAseH per assay. HBV + plasma diluted to the levels shown in HBV- human serum or HBV- serum alone was tested.
Ten ~l of serum were added to an equal volume of 0.1 N KOH and covered with a layer of oil to prevent evaporation. The samples were mixed, heated at 95°C and allowed to cool to room temperature. The samples were brought to 90 ~cl reaction volume with a final concentration of 50 mM Tris acetate pH 7.6, 20.8 mM MgClZ, 5 mM dithiothreitol, 2 mM spermidine hydrochloride, 0.15 GSM each primer, 6.25 mM GTP, 6.25 mM ATP, 2.5 mM UTP, 2.5 mM CTP, 0.2 mM each dTTP, dATP, dGTP, dCTP and 13 U of AMV
reverse transcriptase. The samphs were mixed and heated at 37°C for 12 minutes, then heated to 95°C and cooled to room temperature. Thirteen units of RT and 100 U of T7 RNA polymerase were added arid the reactions were incubated for three hours at 37°C. Twenty-five ~Cl of each reaction was assayed.
The data show that there is a narrow optimum range of concentration of E. coli RNAse H centering around 0.25 U

E. coli RNAse H per reaction for this system. Even though E. coli RNAse H is difficult to use, some added RNAse H
activity was beneficial in 'this experiment.
Table 11 Moles RNAseH RLU observed Target (Units) 5 x 10 2~ 0 567809 5 x 10 ZZ 18041 105 x 10 z~ 2938 5 x 10 Z~ 0.25 1153366 5 x 10 ZZ 732109 155 x 10 z3 5566 5 x 10 z~ 0.5 1001904 5 x 10 zz 29596 205 x 10 z~ 1793 5 x 10~2~ 1.0 610485 5 x 10 z2 13026 5 x 10 23 4062 Examt~le 12 Next the amount of E. coli RNAse H needed for optimal amplification of an HIV primer pair was determined in the 30 presence or absence of a lysate containing 8 ~,g of human DNA. 2 x 1098 moles of viral target were present in each reaction. DNA target was mixed with 50 pmol of each primer in 40 mM Tris HCl pH 8.3, 25 mM NaCl, 20.8 mM MgCl2, 5 mM dithiothreitol, 2 mM spermidine hydrochloride, and 35 nucleotide triphosphates as described for Table 11, heated to 95°C and cooled to room temperature. Thirteen units of AMV reverse transcriptase were added and the reaction 2~~~~~~

heated to 42°C for 12 minutes, to 95°C and cooled again to room temperature. Thirteen units of AMV reverse transcriptase and 100 units of T7 RNA polymerase were added and the reactions heated to 37 °C for 3 . 5 hours prior to assay.
Table 12 Lysate RNAse, H RLU

- 0 U 8,400 - 0.5 239,000 - 1.0 468,000 - 1.5 498,000 - 2.0 439,000 - 3.0 ~ 20,100 - 4.0 5,806 + .0 1,667 + 0.5 924 ~

+ 1.0 6,635 2p + 1.5 ~ 579 + 2.0 13,400 + 3.0 17,800 + 4.0 9,152 These results illustrate that E. colt RNAse H levels have to be carefully controlled as too much _E. coli RNAse H was detrimental t~ the amplification. Additionally, the optimal concentration was altered by the presence of non-specific human DNA and the inhibition by human DNA was significant at all RNAse H levels.
Example 13 We investigated the effect of E. coli RNAse H on amplification of a second region referred to as HIV region 2. The following data demonstrate that E. coli RNAse H
enhances amplification within a narrow concentration range. The HIV region 2 primers were amplified as described for Table 12 in the presence of different concentrations of E. coli RNAse H. Ten microliters of each reaction were assayed and dilutions were made when necessary. Signals were compared to a standard curve to determine the amount of target made.
RNAse H pmole target pmole product Amplification observed - 1.67 x10~~~ 2.14 1.3 x 10~~

0.2 U 1.67 x10 ~ 0.17 1.0 x 109 0.4 U 1.67 X10 ~~ 0.18 1.1 X 109 1.2 U 1.67 x10~~~ 0.012 7.6 x 10~

- 1.67 x108 16.0 9.6 x 108.

0.2 U 1.67 x108 20.6 1.2 x 109 0.4 U 1.67 x108 0.14 8.5 x 106 1.2 U 1.67 x108 0.15 9.0 x 106 These data show that amplif ication can be achieved without the addition of E. coli RNAse H, contrary to the assertions of Guatelli, et al., 87 PNAS 1874-1878.
We investigated using E. coli RNAse H with MMLV
reverse transcriptase in several target regions. Reac-tions were done in the presence or absence of 8 ~.g human DNA using conditions described for Example 12, with a 3 hoax autocatalysis step. Primer sets from HIV regions 1, 3 and 4 were tested. The amount of viral template used was selected to give RLU in the linear range of the hybridization assay. riLNiLV reverse transcriptase was used at 400 U during initiation, 800 U for amplification. 400 U of T7 RNA polymerase were included, and l U of E. coli RNAse H was added as indicated: Values presented under the column headings, +RNAse H, -RNAse H, are RLUs obtained from assay of 10 dal of the reactions.

Table 15 Target Human Region Moles DNA +RNAse -RNAse Target H H

HIV region 1 2 x 10~z~ - 54,900 137,000 fiIV region1 2 x 10 Z~ 8 ~Cg 15, 100 13, 800 HIV region 3 2 x lOZ - 96,100 391,000 HIV region 3 2 x 102 8 ~Cg 124, 000 246, 000 HIV region 4 2 x 102 - 20,400 107,000 HIV region 4 2 x 10 Z' 8 ~Sg 56, 000 8 , 800 In the presence of DNA, E. coli RNAse H apparently stimulated amplification directed by the HIV region 4 primers. In most cases we have tested, amplification using MMLV reverse transcriptase alone is at least as good as when MMLV reverse transcriptase is used with E. coli RNAse H. E. coli RNAse H is not requixed for efficient amplification, contrary to the assertions of Guatelli, et al., 87 PNAS 1874-1878.
Sequence Specificitv of Reverse Transcri~tase We also have discovered that some primer sets work best with AMV reverse transcriptase while others work best with MMLV reverse transcriptase or with one of the reverse transcriptases and added E. aoli RNAse H. The observed degree of variability in amplification efficiency with small changes in promoter primers and primers or source of RNAse H supports our proposed mechanism. We sat forth below our detailed data in support of these findings.
MMLV reverse transcriptase has been cloned, and is commercially available from BRL (Bethesda Research Labs), U.S. Biochemicals and others in a very concentrated farm (greater than 300 units per ~,1). It should be noted that comparable DNA synthetic activity on natural nucleic acid templates is obtained with approximately 10-fold greater unit concentration of MMLV reverse transcriptase compared to AMV reverse transcriptase. Lack of comparability in unit activity is due to the fact that the enzymes show different relative activities when tested with homopolymer templates (used in the unit activity assay) and heteropolymeric nucleic acid templates. We tested the use of MMLV reverse transcriptase at various levels in our amplification reactions. Examples of these results are shown in the tables below. In the AMV reverse transcriptase samples, reverse transcriptase was used at 14 U during the initiation step and 56 U during the amplification step. The amount of MMLV reverse transcriptase was titrated for both the initiation and the amplification steps. The incubation conditions used were as described for Example 12 except that 15 pmol of each HIV region 2 primer was used and 25 mM KCl replaced the NaCl. T7 polymerase was used at 400 U during the amplification. The following table shows performance in the presence or absence of 8 ug human DN,A. Columns headed with the designation AMV or MMLV show the results of amplifications performed with AMV reverse transcriptase or MMLV reverse transcriptase, respectively. The numbers refer to the number of units used during initiation and autocatalysis, respectively. The values contained within the table are RLUs. Note, that dilutions of the amplification products were not performed and values >200,000 RLU may significantly underestimate the extent of amplification since signal saturation occurs at a level of hybridization target sufficient to give about 250,000 RLU
with the conditions used.

Table 16 Human AMV MMLV MMLV t~'!LV

Moles Target DNA 14/56 400/400 400/600 400/800 0 - 495 470 - 3,800 1.6 x lOZZ - 278,000 77,000 - 5,621 1.6 x 102 - 292,000 278,000 - 269,000 0 + 474 547 488 1,352 1.6 x 10 z + 10,200 62,700 205,000 149,000 Although the sensitivity of amplification directed by MMLV
reverse transcriptase in the absence of human DNA was significantly lower than AMV directed amplification, the MMLV was much more effective in the presence of exogenous DNA.
After observing the high level amplification of the HIV region 2, we tested the other target regions in the presence of human DNA and found that, using AMV reverse transcriptase, E. coli RNAse H was still required for the most effective amplification in these regions. We then tested each target region using MMLV reverse transcriptase, without E. coli RNAse H, to compare amplification performance with reactions containing AMV
reverse transcriptase + E. coli RNAse H. An example of these results for two target regions is shown in the table below. The HIV region 3 and 4 primers were used (50 pmol per reaction) as described for Example 12. In reactions using AMV reverse transcriptase, 14 U was used at initi-ation, 56 U reverse transcriptase + 1 U E. coli RNAse H
were added for amplification. In reactions using MMLV
reverse transcriptase, 400 U was added at initiation and 800 U for amplification. All reactions contained 400 U T7 RNA polymerase during the four-hour amplification incu-bation. Values within the tables are RLU obtained from Homogeneous Protection Assay performed using 10 ~.1 of the amplification reactions. Dilutions of the reactions were performed in some cases before assay.

6s Table 17 Human HIV region 3 HIV region 4 Moles Target DNA AMV MMLV AMV MMLV
0 - 2,049 751 1,184 777 1. 6 x 10 z~ - 70, 800 689 2. 1 x 305,000 10~

1.6 x 10z~ - 510,000 1,869 - -0 + 551 400 1,058 1,182 1.6 x 10 2~ + - - 13,900 16,200 1. 6 x 10-2~ + 706 1, 862 141, 000 154,000 1.6 x 10 ~9 + - - 683, 000 723,000 1. 6 x 10-~$ + 10, 800 115, - -As observed in the HIV region 2, in the absence of human DNA, the amplification with MMLV is significantly less than with AMV reverse transcriptase +RNAse H but in the presence of DNA the MMLV directed amplification is at least as good as accomplished by AMV reverse transcriptase-t-RNAse H.42 Example 14 Primers for Second RecZion of HBV g~enome The following experiment was performed with primer sets directed to two regions of the HBV genome. The data show that the primer sets do not amplify to the same extent with AMV RT. The experiment was performed as described for Example 11 using HBV positive plasma diluted in negative serum. Ten microliters of amplification reaction were tested in the hybridisation assay.

Table 18 RLU observed Moles Target FiBV Region 1 HBV Region 4 . 1021 690, 674 8 x 4.8 1022 475,849 73,114 x 4.8 1023 242,452 4,193 x 0 1,417 1,940 These results were confirmed by additional experiments using standard protocols. The region 1 primers consistently gave higher RLU in these experiments.
In contrast, when 800 U of MMLV enzyme were used to amplify the same two primer pairs, the opposite effect was seen as shown below.
Table 19 RLU observed Moles Target Region 1 Region 2 9.6 x 102 37,278 1,067,951 9.6 x IOzZ 1,858 40,826 0 1,010 1,646 In this experiment, each reactian contained 5 ~1 serum.
Thus, the amplification potential of each primer pair was influenced by the reverse transcriptase present during amplification. Factors such as the availability of the template seguences, ability of the primers to hybridize specifically, and efficiency of RNA synthesis should not be affected significantly by the type of reverse transcriptase present. Factors such as RNAse H
specificity and activity and DNA polymerizing activity could be affected.

The following data illustrates that promoter-primer combinations used in the appropriate conditions can be designed to increase amplification.
Table 21 This experiment was performed with HBV region 2 primers as described for Table 11 except that the entire amplification reaction was analyzed by hybridization.
Target Molecules Moles Target RLU Observed 1200 2 x 10-2 1, 094, 177 120 2 x 10 Zz 442, 137 12 2 x 1023 24, 053 1. 2 2 x 10~2~ 8, 654 0 0 1,828 Example 15 Comparison of AMV reverse transcriptase and MMLV reverse transcri~tase.
The following experiment compared amplification of primers for a BCL-2 chromosomal translocation major human chromosomal breakpoint t(14;18) found in patients with CML
using MMLV (300 units) or AMV (39 units). The effect of E. coli RNAse H was evaluated with each enzyme.
Amplifications were performed as described for Example 12 except that 24 mM MgCl2 and 50 pmol each pramer were used.
In reactions containing lysate, 4 ~g of DNA from white blood cells was present. All reactions contained 300 units T7 RNA polymerase and 10 amol of input double-stranded DNA target.
Table 22 RT Lysate O Units 0.5 Units 1.0 Units 2.0 Units RNAse H RNAse H RNAse H RNAse H
AMV - 108,018 2,035,377 -+ 44,485 204,894 165,972 136,647 MMLV - 3,015,948 2,224,666 - -+ 3,070,136 2,714,257 767,218 105,845 ~o~oo~~
The results show that MMLV and AMV RT do not amplify this primer set to the same extent, particularly in the absence of E_. coli RNAse H. E. coli RNAse H added to reactions containing AMV reverse transcriptase markedly improved amplification; this indicates 'that the RNAse activity was limiting in 'these reactions. In contrast, E. coli RNAse H did not enhance amplification when added to reactions containing MMLV RT. The data also confirms a point already made concerning the ability of MMLV RT to sustain significant amplification in the presence of large amounts of nonspecific human DNA.
C. One Primer was not 'Cncorporated into Full Lenqth Product One of our most important findings is that the primer of the same polarity as the major RNA species was not detectably incorporated into full length DNA product. To demonstrate this, individual primers were terminally labeled with 32P, and the incorporation of each primer into DNA products was examined by polyacrylamide gel electrophoresis. We initially expected both primers to be incorporated into full length DNA products. However, the primer containing the promoter. was not observed in full length DNA product. In fact, it remained quite small.
These results were confirmed with a number of different targets and primer sets. Our method is explained below.
To identify the species of cDNA accumulated during autocatalysis, primers were 32P-labeled at the 5° end with T4 polynucleotide kinase and spiked into amplification reactions. The following examples show that cDNA of one polarity is preferentially accumulated during amplification, and that the accumulated strand is always complementary to the predominant RNA species synthesized during autocatalysis. Figure 5 shows the result of incorporation of ~ZP-labeled HIV region 2 primers during amplification. This primer set results in synthesis of a 2U2~9~g 214 base RNA of the (+) sense. The primer complementary to the RNA was incorporated into two major bands of 235 and 214 bases when target sequences were present (lane 3).
No full length fragments were seen in the absence of target (lane 4). The 214 base fragment represents cDNA
equal in length to the RNA while the 235 base fragment is equal in length to the RNA + 21 bases of the T7 promoter sequence. In contrast, the promoter primer was not observed in full length DNA product in the presence or absence of target (lanes 1 and 2 respectively).
Lanes 5-8 of Figure 5 show the result of incorporation of 3ZP-labeled HBV region 1 primers during amplification. These are known as T7pro~' and T7pro-. This primer set is capable of producing 132 base RNA of two polarities but the (+) strand RNA predominates. T7pro-, which is complementary to the predominant RNA, was incorporated into fragments of 132 bases and 153 bases consistent with our proposed mechanism (lane 7 (+) target, lane 8, (-) target) . The 153 base fragment is equal in length to the RNA + 21 bases of the T7 promoter sequence of the T7 pro+. In contrast, 3ZP-labeled T7pro+ primer was not incorporated into fragments of either length (lane 5 (+) target, lane 6, (-) target).
The reactions anaylzed by get electrophoresis were also analyzed by HPA to determine if cDNA of the same polarity as the predominant RNA could be detected by another method. Plus and minus strand probes were used to determine the relative ratio of strands made, and a portion of each reaction was treated with RNAse A to determine the ratio of RNA to DNA. The results are set forth below.

Table 23 Probe Polarity et RLU RLU
Primer s No treatment RNase A

(-) *HIV Region 2 679,182 1037 *HIV Region 2 453,675 1464 (+) *HIV Region 2 32,922 1,094,249 *HIV Region 2 39,494 655,595 (-) *HBV Region 1 567,110 4,854 HBV Region 1 671,656 4,210 (+) *HBV Region 1 56,550 303,160 HBV Region 1 77,983 450,332 * = (+) strand primer labeled with 3zP, others, (-) strand primer labeled with 32P.
These results show that the amplifications worked well, even when full length product was not observed with the promoter primer. These results correlate with what was observed in the previous study, that is, most of the signal observed with one sense probe'is from RNA, and the complementary strand signal is as expected, from DNA.
This was true even far the HBV region 1 primer set which should have made RNA of bath polarities.
D. Confirmation of Mechanism Showincx that the Enzyme Cuts RNA of the RNAlDNA Hybrid at Specific Loci Based upon the experiments and observations reported hereinabove, the mechanism for the amplification systems that takes into account probable sequence specificities in the RNAse H is depicted in Fig. 4. In support of this mechanism, since RNAse H sequence specificity is a key element, it is necessary to show that indeed the enzymes cut the RNA of an RNA:DNA hybrid at specific locations.
Furthermore, the locations of the cut sites needed to be in specific regions according to the model in order for good amplification to be obtained. To examine this question, an RNA: DNA hybrid was prepared that contained the RNA that would be generated from a known target and primer set combination. The RNA was labeled with 32P, incubated with AMV reverse transcriptase (containing its associated RNAse H activity), and the reaction products were analyzed by polycryamide gel electrophoresis. The results were entirely consistent with the new reaction mechanism, namely, the fragment size indicated that several small pieces were generated and that these were produced by cuts near one or both ends. The interior region of the molecule was not cut. This experiment confirmed that the enzyme has sequence or structural specificity under the reaction conditions used.
Further experiments were performed to determine where the cuts occurred since the proposed mechanism requires that multiple cuts occur in the region binding the promoter-containing primer. By labeling the termini of the RNA individually and analyzing the digestion products, it was demonstrated that the cuts were made only at the 5' end of the RNA. This also is consistent with the proposed mechanism.
Example 16 Figure 6 shows that the RNAse H activities from AMV, MMLV and _E. coli cut at specific RNAse H cleavage sites.
The arrows in the figure indicate the position of full-length RNA.
Figure 6 shows the result of an experiment in which HIV region 2 RNA was internally labelled with 32P, hybridized to a single-stranded target sequence and nicked with RNAse H from AMV for 45 minutes (lane 2), MMLV for 5, 15, 45 or 120 minutes (lane 3-6) or E. coli RNAse H for 5 minutes (lane 7). The sizes of fragments produced were discrete and different witty each enzyme, indicating specificity of cleavage of the enzymes and varied specificity among enzymes with this template. The most rapid degradation was observed in the presence of E. coli RNAse H, indicating less specificity or greater activity of cutting with this enzyme.
Figure 6b shows the results of hybridization of HBV
region 1 RNA to a synthetic target sequence, followed by nicking with RNAse H from AMV reverse transcriptase for 5, 30, 60 or 120 minutes (lanes 2-5) or F. coli for 1, 3, 15 or 60 minutes (lanes 6-9). Different sized fragments were produced with the two enzymes, indicating specificity of cleavage. When the HBV RNA was labeled on the 3' terminus and nicked with AMV reverse transcriptase, the same sized fragments were observed, indicating that the cleavage sites were near the 5' end of the RNA.
These data indicate that specific sites are cleaved with RNAse H from AMV, MMLV and E. coli, and that at least some sites are different with the three enzymes. The presence of specific sites within the region to be amplified allows the RNA in an RNA:DNA hybrid to be cleaved, allowing autocatalysis to occur efficiently.
Primers designed using out site information show improved amplification efficiency. This is consistent with our observations that certain primer sets amplified to different extents depending on the source of RNAse H.
E. Identification of MMLV and AI~IV RNAse I-I eut sites.
Example 17 To identify sites digested by A1~IV RNAse H, RNA was hybridized with a complementary sequence and nicked with AMV RNAse H. Following denaturation, a primer complementary to the 3' end of the region to be sequenced was hybridized and extended in the presence of dideoxynucleotides by the Sanger sequencing method.
Termination of cDNA synthesis, idicating cleavage of RNA, was observed at the following sites for the HBV RNA:
5° GGGAGAGGUUAUCGC*UGGA*UGUGUCUGCGGCGUUUUAUCA*UAU
UCCUCUUCA*UCCUG...3' To identify sites digested by MMLV RNAse H, RNA was hybridized with a complementary sequence and nicked with MMhv RNAse H. Following denaturation, a primer complementary to the 3' end of the region to be sequenced was hybridized and extended in the presence of dideoxynucleotides by the Sanger sequencing method.
Termination of cDNA synthesis was observed at the following sites for the HBV RNA:
5'_GGGAGAGGUUAUCGC*UGGA*UGUGUCUGCGGC*GUUUUAUCA*
UAUUCCUCUUCAUCCUGC*UGCUAUGCCUCA*UCUUC...-3' The following sites were identified for a second HBV RNA
sequence:
5'_GGGAGACCCGAGAU*UGA*GAUCUUCUGCGAC
GCGGCGAU*UGA*GAUCUGCGUCU*GCGAGGCGAGGGAGU*UCU*UCUU*CUA
GGGGACCUGCCUCGGUCCCGUC*GUCUA...3' The following sites were identified for an HIV RNA
sequence:
5' GGGAGACAAA*UGGCAGUA*UUCAUCCAC
AAUUUUAAAAGAAAAGGGGGGAUUGGGGGGUA
CAGUGCAGGGGAAAGAAUAGUAGACAUAAUAGC*AACAGACA
UAC*AAACUAAAGAAUUACAAAAACAAAUUAC*AAAAAUUCAA
AAUUUUCGGGUUUAUUACAGGGAC*AGC*AGAAA...3' Most of the cleavage sites occurred near the dinucleotides CA or UG. The method used for detecting cleavage sites only identified sites which accumulated during the cleavage reaction. It is expected that additional sites could be cleaved which were not recognized by the method used.
F. Primers for Amplification Systems Based on findings that the various RNAse H enzymes have sequence specificity, we have tested various primer/target combinations and attempted to optimize their performance in amplification systems. Data obtained to date indicates that the piece size of the RNA fragments produced is relatively large and that the fragments probably do not spontaneously dissociate from the duplex.
This is not unexpected since work with AMV reverse tran--scriptase copying AMV RNA or poliovirus RNA showed that ~~~g ~$
the RNA fragments that were produced by the RNAse H were used by the enzyme to prime the synthesis of cDNA from the initially synthesized cDNA strand.
If the RNAse H enzymes have sequence specificity, the amplification reaction proceeds as follows (beginning with the RNA intermediate in the reaction):
The primer complementary to the major RNA species produced during amplification binds at the 3' terminus of the RNA. Since the concentration of primer is high in the reaction, excess primer produces RNA: DNA duplexes which may be cut by the RNAse H activity before being able to initiate synthesis. Therefore, it is preferable that the primer binding region does not contain a large number of sequences recognized by the RNAse H enzyme used in the reaction.
As cut sites occur frequently, it may not be practical in some cases to design an RNA complementary primer without recognized cut sites; in such cases, the cut sites should be as near the 5' terminus as possible to allow the 3' terminal portion of the primer to remain .
annealed to the RNA.
upon extension of the primer by a suitable DNA
polymerise, the binding site for the second primer, which contains the RNA polymerise promoter, must now be exposed.
It is sufficient to remove only a portion of the RNA to allow nucleation and zippering of the primer as it hybridizes to the cDNA, to allow reverse transcriptase mediated binding of the primer and initiation of synthesis, or merely to nick the RNA so that the RNA
fragment that results may be displaced. Since our data show relatively large pieces of RNA are made and that the promoter containing primer is not incorporated into full~-length DNA, the following events can occur:
1. There is sufficient nicking of the RNA to permit binding of the promoter-primer. Whether a nick in the appropriate place simply produces an RNA fragment sufficiently small to melt off and expose the primer binding site or a portion thereof or whether a nick allows an unwinding activity associated with one or more of the enzymes to displace the RNA fragment is not known at this time.
2. The cDNA 3' terminus is extended by the reverse transcriptase to make the promoter region double-stranded DNA.
3. RNA is synthesized from the complex thus made.
This complex would consist of a cDNA bound to RNA and containing a double-stranded DNA promoter.
Thus, there must be a sequence recognized by the RNAse H activity present in the reaction somewhere in or near the binding site for the primer containing the RNA
polymerase promoter.
In some applications, it may also be desirable to not have RNAse H recognition sites within the target sequence itself. Sites within the target may be cleaved and used to produce RNA primers for synthesis of double-stranded cDNA regions. It may be preferable to eliminate the possibility of this enzymatic activity.
New primer sets were designed based upon the model and the RNAse H sequence specificity information that we have obtained. Our design criteria are as follows:
Fox the T7 promoter-primer:
1) The primer region should have one or more cut sites per 20 bases.
2) The primer region should have a cut site near the 5' end.
3) The primer region should have a cut site near the 3' end and possibly a partial site at the 3° end.
4) The primer length should be a18 bases.
5) The Tm est. should be about 55-65°C.
For the other primer:
1) The primer should have few or no RNAse H cut sites.
2) Any cut sites in the primer should be near the 5' end.

3) The primer length should be about 18-28 bases in length.
4) The Tm est. should be about 55-65°C.
Significantly better synthesis was obtained from primer sets designed using these criteria and knowledge of the mechanism and sequence specificities. This shows the utility of the invention in making possible the design of functional primer sets for specific target regions. These are explained more fully below.
Example 18 Our findings regarding RNAse H specificity have been used to design efficient promoter-primer combinations.
Prior art methods simply nonselectively attached promoters to primer sequences. We have been able to design and optimize promoter-primer combinations to increase the yield of amplified product. The following experiment shows that small changes in promoter-primer sequence result in large changes in amplification efficiency.
The following examples show primers from similar regions which were compared for RIdAse H cleavage sites and GP-III amplification efficiency. In each example, duplicate amplifications were performed using common reagents except for the primers being tested.
1. non promoter primers.
In the first example, the nonpromoter primer site for the CML major t(14; 18) breakpoint amplification region was moved 15 bases, resulting in a reduction in the number of putative RPlAse H cut sites from 4 to 2, assuming a 4 base recognition sequence or from 5 to 2 assuming a 3 base recognition sequence. The reaction was performed as described for Example 15 except that 2.5 mm ATP, 16.5 mm MgCl2 and 50 mM Kcl were included. This change in primer sequence had a dramatic positive effect on amplification efficiency. In the second case, an intentional mismatch was placed internally in the non promoter primer of HBV
region 1 to remove the only putative RNAse H cut site, assuming a 4 base recognition site. In the case of a 3 base cut site, one skilled in the art would recognize that the mismatch removed the cut site nearest the 3' end.
This change also had a definitive positive effect on amplification efficiency. The data demonstrate that two methods, changing the position of the primer, or inclusion of mismatches, can be used to enhance amplification.
Presumably, removal of RNAse H cut sites from the non-promoter primer results in more efficient priming of cDNA
synthesis during autocatalysis.
Sequence RLU
Example 1 GGAGCTGCAGATGCTGACCAAC 78,880 GACCAACTCGTGTGTGAAACTCCA 2,552,333 Example 2 TCCTGGAATTAGAGGACAAACGGGC 57,710 TCCTGGAATTAGAGGATAAACGGGC 518,695 2. promoter-primers. The following examples show promoter primers which come from similar regions but which differ in the number of putative RNAse H cut sites. In the first case, the two promoter primer sites for the HIV
region 5 axe displaced by 10 bases, changing the number of putative RNAse H cut sites from two to three, assuming a four base recognition site, or from 3 to 5 assuming a 3 base recognition site. This has a positive effect on amplification efficiency. In tlxe second case, a sequence containing putative RNAse H cut sites was inserted upstream of the promoter primer for the major breakpoint t(14); 18) translocation, and one mismatch to the target was included to generate a cut site within the primer region. This also had a positive effect on amplification efficiency. This demonstrates that insertion of RNAse H
cut sites in the promoter primer can be used to enhance amplification efficiency. Presumably, inclusion of RNAse H cut sites assists in RNA strand displacement, increasing the efficient of copying of the promoter region, thereby resulting in mare efficient autocatalysis.

Primer name RLU
Example 3 A 45,466 B 908,147 Example 4 C 64,601 D 2,946,706 Sequences of the primers above are:
Primer A:
AATTTTAATACGACTCACTATAGGGAGAAATCTTGTGGGGTGGCTCCTTCT-3' Primer B:
AATTTAATACGACTCACTATAGGGAGAGGGGTGGCTCCTTCTGATAA TGCTG-3' Primer C:
ATTTAATACGACTCACTATAGGGAGACGGTGACCGTGGTCCCTTG-3' Primer D:
TAAATTAATACGACTCACTATAGGGAGATCAGTTACAATCGC
TGGTATCAACGCTGAGCAGACGCTGACCGTGGTCCCTTG-3' In the above examples, removal of RNAse H cut sites from the non-promoter primer resulted in enhanced amplification, even if the removal of the cut site involved the incorporation of a mismatch to the original target. Design of the promoter-containing primer to include additional RNAse H cut sites also enhanced amplification, again, even if the incorporation of cut sites involved inclusion of mismatches to the original target. The number, distribution, and position of putative RNAse H cut sites determine, in part, the usefulness of a given primer.
Improvement of amplification by inclusion of intentional mismatches or insertion of sequences between the promoter and primer are nonobvious improvements to the amplification method.
In a preferred emb~diment of the present invention, the RNA target sequence is determined and then analyzed to determine where RNAse H degradation will cause cuts or removal of sections of RNA from the duplex.
Experiments can be conducted to determine the effect of the RNAse degradation of the target sequence by RNAse H
present in AMV reverse transcriptase and MMLV reverse ~a~~g~~

transcriptase, by E. coli RNAse H or by combinations thereof , In selecting a primer, it is preferable that the primer be selected so that i~t will hybridize to a section of RNA which is substantially nondegraded by the RNAse H
present in the reaction mixture. If there is substantial degradation, the outs in the RNA strand in the region of the primer may stop or inhibit DNA synthesis and prevent extension of the primer. Thus, it is desirable to select a primer which will hybridize with a sequence of the RNA
target, located so that when the RNA is subjected to RNAse H, there is no substantial degradation which would prevent formation of the primer extension product.' The site for hybridization of the promoter-primer is chosen so that sufficient degradation of the RNA strand occurs to permit removal of the portion of the RNA strand hybridized to the portion of the DNA strand to which the promoter-primer will hybridize. Typically, only portions of RNA are removed from the RNA:DNA duplex by RNAse H
degradation and a substantial part of the RNA strand .
remains in the duplex. An RNA:DNA duplex containing a double-stranded DNA promoter results.
,,

Claims (54)

1. A method of synthesizing multiple copies of a target nucleic acid sequence, which comprises:
(a) treating a nucleic acid which comprises an RNA target sequence with a first oligonucleotide which comprises a first primer which has a complexing sequence sufficiently complementary to the 3'-terminal portion of the target sequence to complex therewith and which optionally has a sequence 5' to the complexing sequence which includes a promoter for an RNA polymerase, under conditions whereby an oligonucleotide/target sequence complex is formed and DNA
synthesis may be initiated;
(b) extending the primer in an extension reaction using the target as a template to give a first DNA primer extension product complementary to the RNA target;
(c) separating the first DNA primer extension product from the RNA target using an enzyme which selectively degrades the RNA target;
(d) treating the first DNA primer extension product with a second oligonucleotide which comprises a primer or a splice template and which has a complexing sequence sufficiently complementary to the 3'-terminal portion of the first DNA extension product to complex therewith, under conditions whereby an oligonucleotide/first DNA extension product complex is formed and DNA synthesis may be initiated, provided that if the first oligonucleotide does not have a promoter, then the second oligonucleotide is a splice template which has a sequence 5' to the complexing sequence which includes a promoter for an RNA polymerase;

(e) extending the 3'-terminus of either the second oligonucleotide or the first primer extension product, or both, in a DNA extension reaction to produce a template for an RNA polymerase; and (f) using the template of step (e) to produce multiple RNA copies of the target sequence using an RNA
polymerase which recognizes the promoter sequence whereby if the first oligonucleotide comprises a promoter for a RNA
polymerase and if the polymerase uses the promoter in the first oligonucleotide, the multiple RNA copies will be complementary to the RNA target sequence, wherein the method is conducted under conditions of constant temperature and wherein a reverse transcriptase comprising RNase H activity is used in the method and no other enzyme comprising RNase H activity is used in the method.
2. The method according to claim 1, which further comprises using the oligonucleotides and RNA copies to autocatalytically synthesize multiple copies of the target sequence.
3. A method of synthesizing multiple copies of a target nucleic acid sequence, which comprises:
(a) treating a target nucleic acid which comprises an RNA target sequence with a first oligonucleotide which comprises a first primer which has a complexing sequence sufficiently complementary to the 3'-terminal portion of the target sequence to complex therewith and a sequence 5' to the complexing sequence which includes a promoter for an RNA polymerase, under conditions whereby an oligonucleotide/target sequence complex is formed and DNA
synthesis may be initiated;

(b) extending the first primer in an extension reaction using the target as a template to give a first DNA
primer extension product complementary to the RNA target;
(c) separating the first DNA primer extension product from the RNA target using an enzyme which selectively degrades the RNA target;
(d) treating the first DNA primer extension product with a second oligonucleotide which comprises a second primer which has a complexing sequence sufficiently complementary to the 3'-terminal portion of the first DNA
extension product to complex therewith, under conditions whereby an oligonucleotide/first DNA extension product is formed and DNA synthesis may be initiated;
(e) extending the 3'-terminus of the second primer in a DNA extension reaction to give a second DNA
primer extension product, thereby producing a template for an RNA polymerase; and (f) using the template of step (e) to produce multiple RNA copies of the complement of the RNA target sequence using an RNA polymerase which recognizes the promoter sequence wherein the method is conducted under conditions of constant temperature and, wherein a reverse transcriptase comprising RNase H
activity is used in the method and no other enzyme comprising RNase H activity is used in the method.
4. The method according to claim 3, wherein the second primer has a sequence 5' to the complexing sequence which includes a promoter for an RNA polymerase.
5. The method according to claim 3 or 4, which further comprises using the primers and RNA copies to autocatalytically synthesize multiple copies of the target sequence.
6. The method according to claim 5, wherein the second primer has a sequence 5' to the complexing sequence which includes a promoter for an RNA polymerase.
7. The method according to claim 3, further comprising:
(g) treating an RNA copy from step (f) with the second primer under conditions whereby a second primer/RNA
copy complex is formed and DNA synthesis may be initiated;
(h) extending the 3'-terminus of the second primer in a DNA extension reaction to give a second DNA
primer extension product which is complementary to the RNA
copy;
(i) separating the second DNA primer extension product from the RNA copy using an enzyme which selectively degrades the RNA copy;
(j) treating the second DNA primer extension product with the first primer under conditions whereby a first primer/second DNA primer extension product complex is formed and DNA synthesis may be initiated;
(k) extending the 3'-terminus of the first primer in a DNA extension reaction to give a first DNA primer extension product and the 3'-terminus of the second DNA
primer extension product, thereby producing a template for an RNA polymerase; and (l) using the template of step (k) to produce multiple copies of the target sequence using an RNA
polymerase which recognizes the promoter.

87~
8. The method according to claim 7, wherein the second primer has a :sequence 5' to the complexing sequence which includes a promoter for an RNA polymerase.
9. The method according to claim 7, further comprising:
(m) using an RNA copy of step (l), repeating steps (g) to (l) to autocatalytically synthesize multiple copies of the target sequence.
8. The method according to claim 9, wherein the second primer has a sequence 5' to the complexing sequence which includes a promoter for an RNA polymerase.
9. The method according to claim 4, further comprising:
(g) treating the RNA copies of step (f) with the first and second primers under complexing conditions;
(h) extending the primers in a DNA extension reaction using the RNA copies as templates to give DNA
primer extension products;
(i) separating the DNA primer extension products from the RNA copies using an enzyme which selectively degrades the RNA copies;
(j) treating the DNA primer extension products with the primers under complexing conditions;
(k) extending the primers in a DNA extension reaction to give a complementary primer extension product, thereby producing templates for an RNA polymerase; and (l) using the templates of step (k) to produce multiple copies of the target sequence using an RNA
polymerase which recognizes the promoter.
10. The method according to claim 9, further comprising:
(m) using an RNA copy of step (l), repeating steps (g) to (l) to autocatalytically synthesize multiple copies of the target sequence.
11. A method of synthesizing multiple copies of a target nucleic acid sequence which comprises:
(a) treating a nucleic acid which comprises an RNA target sequence with a first oligonucleotide which comprises a first primer which has a complexing sequence sufficiently complementary to the 3'-terminal portion of the target sequence to complex therewith, under conditions whereby an oligonucleotide/target sequence complex is formed and DNA synthesis may be initiated;
(b) extending the 3'-terminus of the first primer in an extension reaction using the target as a template to give a DNA primer extension product complementary to the RNA
target;
(c) separating the DNA primer extension product from the RNA target using an enzyme which selectively degrades the RNA target;
(d) treating the DNA primer extension product with a second oligonucleotide which comprises a splice template which has a complexing sequence sufficiently complementary to the 3'-terminus of the primer extension product to complex therewith and a sequence 5' to the complexing sequence which includes a promoter for an RNA

polymerase, under conditions whereby a second oligonucleotide/DNA primer extension product complex is formed and DNA synthesis is initiated;
(e) extending the 3'-terminus of the primer extension product to add thereto a sequence complementary to the promoter, thereby producing a template for an RNA
polymerase;
(f) using the template of step (e) to produce multiple RNA copies of the target sequence using an RNA
polymerase which recognizes the promoter, wherein the method is conducted under conditions of constant temperature and wherein a reverse transcriptase comprising RNase H activity is used in the method and no other enzyme comprising RNase H activity is used in the method.
12. The method according to claim 11, wherein the 3'-terminus of the splice template is blocked.
13. The method according to claim 11, further comprising:
(g) using an RNA copy of step (f), repeating steps (a) to (f) to autocatalytically synthesize multiple copies of the target sequence.
14. The method according to claim 13, wherein the 3'-terminus of the splice template is blocked.
15. The method according to claim 13, wherein in step (e), the splice template acts as a second primer and the 3'-terminus of the splice template is extended in a DNA
extension reaction to give a second primer extension product which is complementary to the first primer extension product.
16. The method according to claim 15, wherein the first primer has a sequence 5' to the complexing sequence which includes a promoter for an RNA polymerase.
17. A method of synthesizing multiple copies of a target sequence which comprises:
(a) treating a single stranded target nucleic acid which comprises a DNA target sequence having a defined 3'-terminus with a first oligonucleotide which comprises a splice template which has a complexing sequence sufficiently complementary to the 3'-terminal portion of the target sequence to complex therewith and a sequence 5' to the complexing sequence which includes a promoter for an RNA
polymerase, under conditions whereby an oligonucleotide/target sequence complex is formed and DNA
synthesis may be initiated;
(b) extending the 3'-terminus of the target to add a sequence complementary to the promoter, thereby producing a template for an RNA polymerase;
(c) using the template of step (b) to produce multiple RNA copies of the complement of the DNA target sequence using an RNA polymerase which recognizes the promoter;
(d) treating an RNA copy of step (c) with a second oligonucleotide which comprises a primer which has a complexing sequence sufficiently complementary to the 3'-terminal portion of the RNA copy to complex therewith, under conditions whereby a second oligonucleotide/RNA copy complex is formed and DNA synthesis may be initiated;

(e) extending the 3'-terminus of the primer in a DNA extension reaction to give a DNA primer extension product which is complementary to the RNA copy;
(f) separating the DNA primer extension product from the RNA copy by using an enzyme which selectively degrades the RNA copy;
(g) treating the DNA primer extension product:
with the splice template under conditions whereby a splice template/DNA primer extension product complex is formed and DNA synthesis may be initiated;
(h) extending the 3'-terminus of the primer extension product in a DNA extension reaction to add a sequence complementary to the promoter, thereby producing a template for an RNA polymerase; and (i) using the template of step (h) to produce multiple RNA copies of the target sequence using an RNA
polymerase which recognizes the promoter, wherein the method is conducted under conditions of constant temperature and wherein a reverse transcriptase comprising RNase H activity is used in the method and no other enzyme comprising RNase H activity is used in the method.
18. The method according to claim 17, wherein the 3'-terminus of the splice template is blocked.
19. The method according to claim 18, further comprising:
(j) using the RNA copies of step (i), repeating steps (d) to (i) to autocatalytically synthesize multiple copies of the target sequence.
20. A method of synthesizing multiple copies of a target nucleic acid sequence which comprises:
(a) treating a single stranded target nucleic acid which comprises a DNA target sequence with a first oligonucleotide which comprises a first primer which has a complexing sequence sufficiently complementary to the 3'-terminal portion of the target sequence to complex therewith and a sequence which includes a promoter for an RNA polymerase, under conditions whereby an oligonucleotide/target sequence complex is formed and DNA
synthesis may be initiated;
(b) extending the primer in a DNA extension reaction using the target as a template to give a first DNA
primer extension product complementary to the DNA target sequence;
(c) separating the first primer extension product from the target;
(d) treating the first primer extension product with a second oligonucleotide which comprises a second primer which has a complexing sequence sufficiently complementary to the 3'-terminal portion of the primer extension product to complex therewith, under conditions whereby a second oligonucleotide/DNA primer extension product complex is formed and DNA synthesis may be initiated;
(e) extending the 3'-terminus of the second primer in a DNA extension reaction to give a second DNA
primer extension product, thereby producing a template for an RNA polymerase;

(f) using the template of step (e) to produce multiple RNA copies of the complement of the DNA target sequence using an RNA polymerase which recognizes the promoter;
(g) treating an RNA copy from step (f) with the second primer under conditions whereby second primer/RNA
copy complex is formed and DNA synthesis may be initiated;
(h) extending the 3'-terminus of the second primer in a DNA extension reaction to give a second DNA
primer extension product complementary to the RNA copy;
(i) separating the DNA primer extension product from the RNA copy using an enzyme which selectively degrades the RNA copy;
(j) treating the second primer extension product with the first primer, under conditions whereby a first primer/second DNA primer extension product complex is formed and DNA synthesis may be initiated;
(k) extending the 3'-terminus of the first primer in a DNA extension reaction to give a first DNA primer extension product and the 3'-terminus of the second primer extension product, thereby producing a template for an RNA
polymerase; and (l) using the template of step (k) to produce multiple copies of the target sequence using an RNA
polymerase which recognizes the promoter, wherein steps (d) through (l) are conducted under conditions of constant temperature and wherein a reverse transcriptase comprising RNase H activity is used in the method and no other enzyme comprising RNase H activity is used in the method.
21. The method according to claim 20, wherein the second primer has a sequence 5' to the complexing sequence which includes a promoter for an RNA polymerase.
22. The method according to claim 21, which further comprises:
(m) using an RNA copy of step (1) repeating steps (g) to (l) to autocatalytically synthesize multiple copies of the target sequence.
23. A method of synthesizing multiple copies of a target nucleic acid sequence which comprises:
(a) treating a single stranded target nucleic acid which comprises a DNA target sequence with a first oligonucleotide which comprises a primer which has a complexing sequence sufficiently complementary to the 3'-terminal portion of the target sequence to complex therewith under conditions whereby an oligonucleotide/target sequence complex is formed and DNA synthesis may be initiated;
(b) extending the 3'-terminus of the primer in an extension reaction using the target as a template to give a DNA primer extension product complementary to the target;
(c) separating the primer extension product from the target;
(d) treating the primer extension product with a second oligonucleotide which comprises a splice template which has a complexing sequence sufficiently complementary to the 3'-terminal portion of the primer extension product to complex therewith and a sequence 5' to the complexing sequence which includes a promoter for an RNA polymerase, under conditions whereby a second oligonucleotide/primer extension product complex is formed and DNA synthesis may be initiated;
(e) extending the 3'-terminus of the primer extension product to add thereto a sequence complementary to the promoter, thereby producing a template for the RNA
polymerase; and (f) using the template of step (e) to produce multiple RNA copies of the target sequence using an RNA
polymerase which recognizes the promoter;
wherein steps (d) through (f) are conducted under conditions of constant temperature and wherein a reverse transcriptase comprising RNase H activity is used in the method and no other enzyme comprising RNase H activity is used in the method.
24. The method according to claim 23, further comprising:
(g) treating an RNA copy of step (f) with the primer under conditions whereby a primer/RNA copy complex is formed and DNA synthesis may be initiated;
(h) extending the 3'-terminus of the primer in a DNA extension reaction to give a second primer extension product;
(i) separating the second primer extension from the RNA copy using an enzyme which selectively degrades the RNA copy;
(j) treating the second primer extension product with the splice template under conditions whereby a second primer extension product/splice template complex is formed and DNA synthesis may be initiated;

(k) extending the 3'-terminus of the second primer extension product to add thereto a sequence complementary to the promoter, thereby producing a template for an RNA polymerase; and (l) using the template of step (k) to produce RNA
copies of the target sequence using an RNA polymerase which recognizes the promoter wherein steps (g) through (l) are conducted under conditions of constant temperature.
25. The method according to claim 24, wherein the 3'-terminus of the splice template is blocked.
26. The method according to claim 24, further comprising:
(m) using an RNA copy of step (l), repeating steps (g) to (l) to autocatalytically synthesize multiple copies of the target sequence.
27. A method for autocatalytically synthesizing multiple copies of a target nucleic acid sequence under conditions of substantially constant temperature, ionic strength and pH which comprises:
(a) combining:
(1) a target nucleic acid which comprises a single-stranded RNA target sequence;
(2) a first oligonucleotide which comprises a first primer which has a complexing sequence sufficiently complementary to the 3'-terminal portion of the RNA target sequence to complex therewith and which optionally has a sequence 5' to the complexing sequence which includes a promoter for an RNA polymerase;

(3) a second oligonucleotide which comprises a second primer or a splice template and which has a complexing sequence sufficiently complementary to the 3'-terminal portion of a complement to the RNA target sequence to complex therewith, provided that if the first oligonucleotide does not have a promoter, then the second oligonucleotide is a splice template which has a sequence 5' to the complexing sequence which includes a promoter for an RNA polymerase;
(4) a DNA-dependent DNA polymerase;
(5) an RNA-dependent DNA polymerase comprising an enzyme which selectively degrades the RNA strand of an RNA:DNA duplex; and (6) an RNA polymerase which recognizes the promoter; and (b) incubating the mixture of step (a) under DNA
priming and nucleic acid synthesizing conditions, which include substantially constant temperature, ionic strength and pH wherein a reverse transcriptase comprising RNase H
activity is used in the method and no other enzyme comprising RNase H activity is used in the method; and whereby if the first oligonucleotide comprises a promoter for an RNA polymerase and if the polymerase uses the promoter in the first oligonucleotide, the multiple RNA
copies will be complementary to the RNA target sequence.
28. The method according to claim 27, wherein the first primer has a promoter.
29. The method according to claim 27 or 28, wherein the second oligonucleotide comprises a second primer which has a sequence 5' to the second primer complexing sequence which includes a promoter for an RNA polymerase.
30. The method according to claim 29, wherein the DNA-dependent DNA polymerase comprises a reverse transcriptase.
31. A method for autocatalytically synthesizing multiple copies of a target nucleic acid sequence under conditions of substantially constant temperature, ionic strength and pH which comprises:
(a) combining:
(1) a target nucleic acid which comprises, an RNA
target sequence;
(2) a primer and a splice template of opposite sense wherein one has a complexing sequence sufficiently complementary to the 3'-terminal portion of the target sequence of the RNA target to complex therewith and the other has a complexing sequence sufficiently complementary to the 3'-terminal portion of the complement of the target sequence to complex therewith and wherein the splice template has a sequence 5' to the complexing sequence which includes a promoter;
(3) a DNA-dependent DNA polymerase;
(4) an RNA-dependent DNA polymerase comprising an enzyme which selectively degrades the RNA strand of an RNA:DNA duplex; and (5) an RNA polymerase which recognizes the promoter of the splice template; and (b) incubating the mixture of step (a) under DNA
priming and nucleic acid synthesizing conditions which include substantially constant temperature, ionic strength, and pH wherein a reverse transcriptase comprising RNase H
activity is used in the method and no other enzyme comprising RNase H is used in the method.
32. A method for autocatalytically synthesizing multiple copies of a target nucleic acid sequence which comprises:
(a) combining:
(1) a nucleic acid which comprises a single stranded DNA target sequence;
(2) a first primer which has a complexing sequence sufficiently complementary to the 3'-terminal portion of the target sequence to complex therewith and a sequence 5' to the complexing sequence which includes a promoter for an RNA polymerase;
(3) a DNA polymerase;
(b) incubating the mixture of step (a) under DNA
priming and synthesizing conditions whereby a primer extension product complementary to the target sequence is synthesized using the target sequence as a template;
(c) treating the reaction mixture of step (b) to cause separation of DNA duplexes;
(d) adding to the reaction mixture of step (c):
(1) a second primer which has a complexing sequence sufficiently complementary to the 3'-terminal portion of the target sequence of the primer extension product to complex therewith;
(2) a DNA-dependent DNA polymerase;

(3) an RNA-dependent DNA polymerase comprising an enzyme which selectively degrades the RNA strand of an RNA:DNA complex; and (4) an RNA polymerase which recognizes the promoter;
wherein a reverse transcriptase comprising RNase H
activity and no other enzyme comprising RNase H activity is used in the method; and, (e) incubating the mixture of step (d) under DNA
priming and nucleic acid synthesizing conditions which include substantially constant temperature, ionic strength and pH.
33. A method of synthesizing multiple copies of a target nucleic acid sequence which comprises:
(a) selecting a primer, complementary to a portion of an RNA target sequence, which complexes with the portion of the RNA target, the portion of the RNA target located such that it remains substantially undegraded, after exposure to degradation by RNase H;
(b) selecting a promoter-primer, complementary to a portion of the DNA obtained by extension of the primer, which complexes with the DNA in an area where substantially all of the complementary RNA is removed from the RNA target sequence/DNA extension product complex by degradation of the RNA; and, (c) combining the RNA target with the primer, promoter-primer, reverse transcriptase having associated RNase H activity and transcriptase and forming multiple copies of the target nucleic acid sequence under conditions of constant temperature.
34. A method of synthesizing multiple copies of a target nucleic acid sequence which comprises:

(a) selecting a primer, complementary to a portion of an RNA target sequence, which complexes with the portion of the RNA target, the portion of the RNA target located such that it remains capable of forming a primer extension product after being exposed to degradation by a selected RNase H;
(b) selecting a promoter-primer, complementary to a portion of the DNA obtained by extension of the primer, which complexes with the DNA in an area where substantially all of the complementary RNA is removed from the RNA target sequence/DNA extension product complex by degradation of the RNA; and (c) combining the RNA target with the primer, promoter-primer, reverse transcriptase having associated RNase H activity and transcriptase and forming multiple copies of the target nucleic acid sequence under conditions of constant temperature.
35. A method of synthesizing multiple copies of a target nucleic acid sequence which comprises:
(a) selecting a primer, complementary to a portion of an RNA target sequence, which complexes with the portion of the RNA target, the portion of the RNA target located such that it remains substantially undegraded after exposure to degradation by RNase H;
(b) selecting a promoter-primer, complementary to a portion of the DNA obtained by extension of the primer, which complexes with the DNA in an area where substantially all of the complementary RNA is removed from the RNA target sequence/DNA extension product complex by degradation of the RNA; and, (c) combining the RNA target with the primer, promoter-primer, reverse transcriptase having associated RNase H activity and transcriptase and forming multiple copies of the RNA and multiple copies of DNA complementary to the RNA without making a substantially equivalent number of copies of DNA of the same polarity as the RNA target sequence under conditions of constant temperature.
36. A method of synthesizing multiple copies of a target nucleic acid sequence which comprises:
(a) selecting a primer, complementary to a portion of an RNA target sequence, which complexes with the portion of the RICA target, the portion of the RNA target located such that it remains capable of forming a primer extension product after being exposed to degradation by a selected RNase H;
(b) selecting a promoter-primer, complementary to a portion of the DNA obtained by extension of the primer, which complexes with the DNA in an area where substantially all of the complementary RNA is removed from the RNA target sequence/DNA extension product complex by degradation of the RNA; and (c) combining the RNA target with the primer, promoter-primer, reverse transcriptase having associated RNase H activity and transcriptase and forming multiple copies of the RNA and multiple copies of DNA complementary to the RNA without making a substantially equivalent number of copies of DNA of the same polarity as the RNA target sequence under conditions of constant temperature.
37. A method of synthesizing multiple copies of a target nucleic acid sequence which comprises:
(a) selecting a primer, complementary to a portion of an RNA target sequence, which complexes with the portion of the RNA target, the portion of the RNA target located such that it remains capable of forming a primer extension product after being exposed to degradation by a selected RNase H;
(b) selecting a promoter-primer, complementary to a portion of the DNA obtained by extension of the primer, which complexes with the DNA in an area where a sufficient amount of the 5 end of the RNA is removed from the RNA
target sequence/DNA extension product complex so as to permit the further extension of the primer extension product DNA to produce a complement to the promoter-primer; and (c) combining the RNA target with the primer, promoter-primer, reverse transcriptase having associated RNase H activity and transcriptase and forming multiple copies of the target nucleic acid sequence under conditions of constant temperature.
38. A method of synthesizing multiple copies of a target nucleic acid sequence, which comprises:
(a) selecting a primer, complementary to a portion of an RNA target sequence, which complexes with the portion of the RNA target, the portion of the RNA target located such that it remains capable of forming a primer.
extension product after being exposed to degradation by a selected RNase H;
(b) selecting a promoter-primer, complementary to a portion of the DNA obtained by extension of the primer, which complexes with the DNA in an area where a sufficient amount of the 5 end of the RNA is removed so as to permit the promoter-primer to complex to the 3' end of the DNA
obtained by extension of the primer; and (c) combining the RNA target with the primer, promoter-primer, reverse transcriptase having associated RNase H activity and transcriptase and forming multiple copies of the target nucleic acid sequence under conditions of constant temperature.
39. A method of synthesizing multiple copies of a target nucleic acid sequence under conditions of constant temperature, which comprises:
(a) treating a nucleic acid which comprises an RNA target sequence with a first oligonucleotide which comprises a first primer which has a complexing sequence sufficiently complementary to the 3'-terminal portion of the target sequence to complex therewith under conditions whereby DNA synthesis may be initiated;
(b) extending the primer in an extension reaction using the target as a template to give a DNA primer extension product complementary to the RNA target;
(c) selectively digesting the 5' end of the RNA
target with an enzyme having RNase H activity;
(d) treating the DNA primer extension product with a second oligonucleotide which comprises a primer or a splice template and which has a complexing sequence sufficiently complementary to the 3'-terminal portion of the DNA primer extension product under conditions whereby DNA
synthesis may be initiated and has a promoter sequence 5' to the complexing sequence;

(e) extending the 3'-terminus of the first primer extension product in a DNA extension reaction to produce a template for an RNA polymerase; and (f) using the template of step (e) to produce multiple RNA copies of the target sequence using an RNA
polymerase which recognizes the promoter sequence;
wherein a reverse transcriptase comprising RNase H
activity and no other enzyme comprising RNase H activity is used in the method.
40. The method according to claim 36, further comprising using the primer, promoter-primer and an RNA copy to autocatalytically synthesize multiple copies of the target sequence.
41. A blocked splice template comprising a first and a second nucleic acid region, wherein the first region is located 3' of the second region and is blocked at its 3'-terminus to inhibit primer extension by a DNA polymerase, and the second region comprises a promoter sequence recognized by an RNA polymerase.
42. A kit for synthesizing multiple RNA transcripts comprising:
a primer able to hybridize to a 3'-terminal portion of a target sequence and be extended to produce a primer extension product containing a complementary target sequence, and a blocked splice template comprising a first and a second nucleic acid region, wherein the first region is located 3' of the second region, comprises a nucleotide sequence able to hybridize to a 3'-terminal portion of a complementary target sequence, and is blocked at its 3'-terminus to inhibit primer extension by a DNA polymerase, and the second region comprises a promoter sequence recognized by an RNA polymerase.
43. The kit of claim 42, further comprising the RNA
polymerase.
44. The kit of claim 43, further comprising a reverse transcriptase.
45. A method for synthesizing multiple RNA transcripts comprising the steps of:
(a) providing a target nucleic acid comprising a target sequence with a first oliganucleotide comprising a primer having a nucleotide sequence able to hybridize to a 3'-terminal portion of the target sequence, whereby an oligonucleotide:target sequence hybride is formed and DNA
synthesis may be initiated, wherein the target: sequence is initially present or produced by a preliminary procedure;
(b) extending the primer in a primer extension reaction using the target sequence as a template to give a DNA primer extension product comprising a complementary target sequence;
(c) making a 3'-terminal portion of the complementary target sequence available for hybridization with a second oligonucleotide, wherein the second oligonucleotide is a blacked splice template comprising a first and a second nucleic acid region, wherein the first region is located 3' of the second region, comprises a nucleotide sequence able to hybridize to the 3'-terminal portion of the complementary target sequence made available, and is blocked at its 3'-terminus to inhibit extension by a DNA polymerase, and the second region comprises a promoter sequence recognized by an RNA polymerase;
(d) hybridizing the second oligonucleotide to the DNA primer extension product;
(e) extending the 3'-end of the DNA primer extension product to form a double-stranded promoter comprising the promoter sequence; and (f) synthesizing multiple copies of RNA
transcripts complementary to the complementary target sequence using the RNA polymerase and DNA priming and nucleic acid synthesizing conditions which include the necessary substrates and buffer conditions for primer extension and production of the RNA transcripts.
46. The method of claim 45, wherein the target nucleic acid is RNA and the step (c) is carried out using an enzyme which selectively degrades the RNA target when present in a RNA: DNA duplex.
47. The method of claim 46, wherein the enzyme is a reverse transcriptase having RNase H activity.
48. The method of claim 47, wherein the reverse transcriptase alone supplies the RNase H activity.
49. The method of claim 48, wherein the method is carried out under essentially constant temperature.
50. A method for synthesizing multiple RNA transcripts comprising the steps of:
(a) combining:
a target nucleic acid comprising the target sequence, wherein the target sequence is initially present or produced by a preliminary procedure;

a first oliganucleotide comprising a primer able to hybridize to a 3'-terminal portion of the target sequence;
a second oligonucleotide which is a blocked splice template comprising a first and a second nucleic acid region, wherein the first region is located 3' of the second region, comprises a nucleotide sequence able to hybridize to a 3'-terminal portion of a complementary target sequence, and is blocked at its 3'-terminus to inhibit extension by a DNA polymerase, and the second region comprises a promoter sequence recognized by an RNA polymerase;
a reverse transcriptase; and the RNA polymerase;
(b) incubating the mixture of step (a) under DNA
priming and nucleic acid synthesizing conditions which include the necessary substrates and buffer conditions for primer extension and production of RNA transcripts; and (c) synthesizing multiple RNA transcripts.
51. The method of claim 50, wherein the target sequence is RNA and the method is carried out under essentially constant temperature.
52. The method of claim 51, wherein the combining step further comprises an enzyme which selectively degrades the RNA strand of an RNA: DNA duplex.
53. The method of claim 52, wherein the enzyme is a reverse transcriptase having RNase H activity.
54. The method of claim 53 wherein the reverse transcriptase alone supplies the RNase H activity to degrade the RNA strand.
CA002020958A 1989-07-11 1990-07-11 Nucleic acid sequence amplification methods Expired - Lifetime CA2020958C (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US37950189A 1989-07-11 1989-07-11
US379,501 1989-07-11
US07/550,837 US5480784A (en) 1989-07-11 1990-07-10 Nucleic acid sequence amplification methods
US550,837 1990-07-10

Publications (2)

Publication Number Publication Date
CA2020958A1 CA2020958A1 (en) 1991-01-12
CA2020958C true CA2020958C (en) 2005-01-11

Family

ID=27008641

Family Applications (1)

Application Number Title Priority Date Filing Date
CA002020958A Expired - Lifetime CA2020958C (en) 1989-07-11 1990-07-11 Nucleic acid sequence amplification methods

Country Status (2)

Country Link
US (3) US5399491A (en)
CA (1) CA2020958C (en)

Families Citing this family (801)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5714380A (en) 1986-10-23 1998-02-03 Amoco Corporation Closed vessel for isolating target molecules and for performing amplification
CA2020958C (en) * 1989-07-11 2005-01-11 Daniel L. Kacian Nucleic acid sequence amplification methods
US6589734B1 (en) 1989-07-11 2003-07-08 Gen-Probe Incorporated Detection of HIV
AU650622B2 (en) * 1989-07-11 1994-06-30 Gen-Probe Incorporated Nucleic acid sequence amplification methods utilizing a transcription complex
US5766849A (en) * 1989-07-11 1998-06-16 Gen-Probe Incorporated Methods of amplifying nucleic acids using promoter-containing primer sequence
US5780219A (en) * 1989-07-11 1998-07-14 Gen-Probe Incorporated Nucleic acid amplification oligonucleotides and probes to human hepatitis B virus
US7009041B1 (en) 1989-07-11 2006-03-07 Gen-Probe Incorporated Oligonucleotides for nucleic acid amplification and for the detection of Mycobacterium tuberculosis
US5545522A (en) 1989-09-22 1996-08-13 Van Gelder; Russell N. Process for amplifying a target polynucleotide sequence using a single primer-promoter complex
US7049102B1 (en) 1989-09-22 2006-05-23 Board Of Trustees Of Leland Stanford University Multi-gene expression profile
US6136535A (en) * 1991-11-14 2000-10-24 Digene Corporation Continuous amplification reaction
US5981179A (en) * 1991-11-14 1999-11-09 Digene Diagnostics, Inc. Continuous amplification reaction
CA2137563C (en) * 1992-06-08 2005-06-28 Thomas B. Ryder Preparation of nucleic acid from mononuclear cells
CA2159103C (en) 1993-03-26 2002-03-12 Sherrol H. Mcdonough Detection of human immunodeficiency virus type 1
US6986985B1 (en) 1994-01-13 2006-01-17 Enzo Life Sciences, Inc. Process for producing multiple nucleic acid copies in vivo using a protein-nucleic acid construct
US20050123926A1 (en) * 1994-01-13 2005-06-09 Enzo Diagnostics, Inc., In vitro process for producing multiple nucleic acid copies
US20110097791A1 (en) * 1999-04-16 2011-04-28 Engelhardt Dean L Novel process, construct and conjugate for producing multiple nucleic acid copies
US6379897B1 (en) * 2000-11-09 2002-04-30 Nanogen, Inc. Methods for gene expression monitoring on electronic microarrays
US5665545A (en) * 1994-11-28 1997-09-09 Akzo Nobel N.V. Terminal repeat amplification method
CA2139070C (en) * 1994-12-23 2010-03-30 Burton W. Blais Method for enhancing detection ability of nucleic acid assays employing polymerase chain reaction
US5705365A (en) * 1995-06-07 1998-01-06 Gen-Probe Incorporated Kits for determining pre-amplification levels of a nucleic acid target sequence from post-amplification levels of product
US5747252A (en) * 1995-06-07 1998-05-05 Gen-Probe Incorporated Nucleic acid probes and amplification oligonucleotides for Neisseria species
US5710029A (en) * 1995-06-07 1998-01-20 Gen-Probe Incorporated Methods for determining pre-amplification levels of a nucleic acid target sequence from post-amplification levels of product
WO1997018334A2 (en) * 1995-11-15 1997-05-22 Gen-Probe Incorporated Nucleic acid probes complementary to human papillomavirus nucleic acid and related methods and kits
EP0914462A4 (en) * 1996-03-18 2002-05-22 Molecular Biology Resources Target nucleic acid sequence amplification
DE69734595T2 (en) 1996-06-28 2006-08-10 Rebio Gen, Inc., Hachioji METHOD FOR DISCOVERING TELOMERASE ACTIVITY
US5876992A (en) * 1996-07-03 1999-03-02 Molecular Biology Resources, Inc. Method and formulation for stabilization of enzymes
EP0941314A1 (en) 1996-08-02 1999-09-15 Bio Merieux Target nucleic acid sequence amplification method
US5837466A (en) * 1996-12-16 1998-11-17 Vysis, Inc. Devices and methods for detecting nucleic acid analytes in samples
US6025133A (en) * 1996-12-30 2000-02-15 Gen-Probe Incorporated Promoter-sequestered oligonucleoside and method of use
ATE426018T1 (en) 1997-04-10 2009-04-15 Stichting Katholieke Univ PCA3, PCA3 GENES AND METHODS FOR USE THEREOF
US6534273B2 (en) 1997-05-02 2003-03-18 Gen-Probe Incorporated Two-step hybridization and capture of a polynucleotide
DE69836012T2 (en) 1997-05-02 2007-04-05 Gen-Probe Inc., San Diego TWO-STEP HYBRIDIZATION AND INJECTION OF A POLYNUCLEOTIDE
US6503709B1 (en) 1997-07-03 2003-01-07 Id Biomedical Corporation Methods for rapidly detecting methicillin resistant staphylococci
US6849400B1 (en) * 1997-07-23 2005-02-01 Gen-Probe Incorporated Methods for detecting and measuring spliced nucleic acids
FR2768743B1 (en) 1997-09-23 2001-09-14 Bio Merieux PROCESS FOR LYSIS OF MICROORGANISM
US20040002079A1 (en) * 1998-02-11 2004-01-01 Gunn Robert B. Sodium-phosphate cotransporter in lithium therapy for the treatment of mental illness
US7090975B2 (en) * 1998-03-13 2006-08-15 Promega Corporation Pyrophosphorolysis and incorporation of nucleotide method for nucleic acid detection
US6235480B1 (en) 1998-03-13 2001-05-22 Promega Corporation Detection of nucleic acid hybrids
US6277578B1 (en) 1998-03-13 2001-08-21 Promega Corporation Deploymerization method for nucleic acid detection of an amplified nucleic acid target
US6703211B1 (en) 1998-03-13 2004-03-09 Promega Corporation Cellular detection by providing high energy phosphate donor other than ADP to produce ATP
US6270973B1 (en) 1998-03-13 2001-08-07 Promega Corporation Multiplex method for nucleic acid detection
US6270974B1 (en) 1998-03-13 2001-08-07 Promega Corporation Exogenous nucleic acid detection
US6268146B1 (en) 1998-03-13 2001-07-31 Promega Corporation Analytical methods and materials for nucleic acid detection
US6312902B1 (en) 1998-03-13 2001-11-06 Promega Corporation Nucleic acid detection
US6391551B1 (en) 1998-03-13 2002-05-21 Promega Corporation Detection of nucleic acid hybrids
DE69936237T2 (en) 1998-05-01 2008-01-31 Gen-Probe Inc., San Diego Stirring device for the fluid content of a container
WO1999058724A1 (en) * 1998-05-08 1999-11-18 Life Technologies, Inc. A method for synthesizing a nucleic acid molecule using a ribonuclease
AU762728B2 (en) * 1998-07-02 2003-07-03 Gen-Probe Incorporated Molecular torches
US6551778B1 (en) 1999-01-28 2003-04-22 Gen-Probe Incorporated Nucleic acid sequences for detecting genetic markers for cancer in a biological sample
DE60043292D1 (en) 1999-02-12 2009-12-24 Gen Probe Inc PROTECTION PROBES
US6582906B1 (en) 1999-04-05 2003-06-24 Affymetrix, Inc. Proportional amplification of nucleic acids
US6235479B1 (en) 1999-04-13 2001-05-22 Bio Merieux, Inc. Methods and devices for performing analysis of a nucleic acid sample
US20060275782A1 (en) * 1999-04-20 2006-12-07 Illumina, Inc. Detection of nucleic acid reactions on bead arrays
EP1923472B1 (en) 1999-04-20 2012-04-11 Illumina, Inc. Detection of nucleic acid reactions on bead arrays
US20030207295A1 (en) * 1999-04-20 2003-11-06 Kevin Gunderson Detection of nucleic acid reactions on bead arrays
US6821770B1 (en) * 1999-05-03 2004-11-23 Gen-Probe Incorporated Polynucleotide matrix-based method of identifying microorganisms
US6235484B1 (en) 1999-05-03 2001-05-22 Gen-Probe Incorporated Polynucleotide probes for detection and quantitation of actinomycetes
WO2000066785A2 (en) 1999-05-03 2000-11-09 Gen-Probe Incorporated Polynucleotide probes for detection and quantitation of bacteria in the family enterobacteriaceae
JP4913282B2 (en) 1999-05-03 2012-04-11 ジェン−プローブ・インコーポレーテッド Polynucleotide probes for detection and quantification of Staphylococcus
US6132997A (en) * 1999-05-28 2000-10-17 Agilent Technologies Method for linear mRNA amplification
US6531300B1 (en) * 1999-06-02 2003-03-11 Saigene Corporation Target amplification of nucleic acid with mutant RNA polymerase
EP1422298B1 (en) * 1999-07-09 2010-01-27 Gen-Probe Incorporated Detection of hiv-1 by nucleic acid amplification
DE60022541T2 (en) 1999-07-23 2006-06-14 Gen Probe Inc POLYNUCLEOTIDE AMPLIFICATION PROCESS
US6864050B2 (en) * 1999-07-30 2005-03-08 Affymetrix, Inc. Single-phase amplification of nucleic acids
US6472156B1 (en) 1999-08-30 2002-10-29 The University Of Utah Homogeneous multiplex hybridization analysis by color and Tm
DE60009323T2 (en) 1999-09-13 2005-02-10 Nugen Technologies, Inc., San Carlos METHODS AND COMPOSITIONS FOR LINEAR ISOTHERMAL AMPLIFICATION OF POLYNUCLEOTIDE SEQUENCES
US6692918B2 (en) 1999-09-13 2004-02-17 Nugen Technologies, Inc. Methods and compositions for linear isothermal amplification of polynucleotide sequences
DK1222266T3 (en) 1999-09-29 2006-07-10 Diagnocure Inc PCA3 messenger RNA in benign and malignant prostate tissue
GB2355791B (en) * 1999-10-28 2004-10-20 Molecular Light Tech Res Ltd Detection of mRNA expression using chemiluminescent labelled probes
US20010031466A1 (en) * 1999-12-02 2001-10-18 Malek Lawrence T. Preparation of sequence libraries from non-denatured RNA and kits therefor
US6902891B2 (en) 1999-12-17 2005-06-07 Bio Merieux Process for labeling a nucleic acid
US6489114B2 (en) 1999-12-17 2002-12-03 Bio Merieux Process for labeling a ribonucleic acid, and labeled RNA fragments which are obtained thereby
US7250252B2 (en) * 1999-12-30 2007-07-31 David Aaron Katz Amplification based polymorphism detection
US20030022318A1 (en) * 2000-01-25 2003-01-30 Epiclone, Inc. Method for thermocycling amplification of nucleic acid sequences and the generation of related peptides thereof
US20050214825A1 (en) * 2000-02-07 2005-09-29 John Stuelpnagel Multiplex sample analysis on universal arrays
US7955794B2 (en) * 2000-09-21 2011-06-07 Illumina, Inc. Multiplex nucleic acid reactions
US7582420B2 (en) 2001-07-12 2009-09-01 Illumina, Inc. Multiplex nucleic acid reactions
US8076063B2 (en) 2000-02-07 2011-12-13 Illumina, Inc. Multiplexed methylation detection methods
ATE441726T1 (en) * 2000-02-23 2009-09-15 Hope City PYROPHOSPHOROLYSIS-ACTIVATED POLYMERIZATION (PAP): APPLICATION FOR ALLEL-SPECIFIC AMPLIFICATION AND SEQUENCE DETERMINATION OF NUCLEIC ACIDS
US7033763B2 (en) * 2000-02-23 2006-04-25 City Of Hope Pyrophosphorolysis activated polymerization (PAP)
US7846733B2 (en) * 2000-06-26 2010-12-07 Nugen Technologies, Inc. Methods and compositions for transcription-based nucleic acid amplification
AU2001271595A1 (en) * 2000-06-26 2002-01-08 Nugen Technologies, Inc. Methods and compositions for transcription-based nucleic acid amplification
US20080242627A1 (en) * 2000-08-02 2008-10-02 University Of Southern California Novel rna interference methods using dna-rna duplex constructs
CA2419649C (en) 2000-09-01 2011-03-08 Gen-Probe Incorporated Amplification of hiv-1 sequences for detection of sequences associated with drug-resistance mutations
US6582920B2 (en) 2000-09-01 2003-06-24 Gen-Probe Incorporated Amplification of HIV-1 RT sequences for detection of sequences associated with drug-resistance mutations
WO2002022890A2 (en) * 2000-09-12 2002-03-21 Gen-Probe Incorporated Compositions, methods and kits for determining the presence of cryptosporidium organisms in a test sample
WO2002029117A2 (en) 2000-10-06 2002-04-11 Nugen Technologies, Inc. Methods and probes for detection and/or quantification of nucleic acid sequences
US6870045B2 (en) 2000-10-23 2005-03-22 Gen-Probe Incorporated Kits for detecting HIV-2
DE60142709D1 (en) * 2000-12-13 2010-09-09 Nugen Technologies Inc METHODS AND COMPOSITIONS FOR GENERATING A VARIETY OF COPIES OF NUCLEIC ACID SEQUENCES AND METHODS OF DETECTING THE SAME
US6794141B2 (en) 2000-12-22 2004-09-21 Arcturus Bioscience, Inc. Nucleic acid amplification
EP1366196A4 (en) * 2001-02-14 2004-07-07 Baylor College Medicine Methods and compositions of amplifying rna
EP1390537B1 (en) * 2001-03-09 2013-11-13 Nugen Technologies, Inc. Methods and compositions for amplification of rna sequences
ES2271232T3 (en) 2001-03-09 2007-04-16 Gen-Probe Incorporated PERFORABLE CAPERUZA.
ATE361996T1 (en) * 2001-03-09 2007-06-15 Nugen Technologies Inc METHODS AND COMPOSITIONS FOR DUPLICATION OF RNA SEQUENCES
US7338805B2 (en) 2001-05-04 2008-03-04 Bio Merieux Labeling reagents, methods for synthesizing such reagents and methods for detecting biological molecules
GB0111275D0 (en) * 2001-05-09 2001-06-27 Secr Defence Analytical method and kit
US8137911B2 (en) * 2001-05-22 2012-03-20 Cellscript, Inc. Preparation and use of single-stranded transcription substrates for synthesis of transcription products corresponding to target sequences
WO2004059289A2 (en) * 2001-05-22 2004-07-15 Epicentre Technologies Target-dependent transcription using deletion mutants of n4 rna polymerase
CA2451756A1 (en) * 2001-06-28 2003-01-09 Chiron Corporation Diagnostic assays for parvovirus b19
US9261460B2 (en) 2002-03-12 2016-02-16 Enzo Life Sciences, Inc. Real-time nucleic acid detection processes and compositions
US20040161741A1 (en) * 2001-06-30 2004-08-19 Elazar Rabani Novel compositions and processes for analyte detection, quantification and amplification
US9777312B2 (en) * 2001-06-30 2017-10-03 Enzo Life Sciences, Inc. Dual polarity analysis of nucleic acids
EP2246438B1 (en) 2001-07-12 2019-11-27 Illumina, Inc. Multiplex nucleic acid reactions
TWI335938B (en) * 2001-08-15 2011-01-11 Rna replication and amplification
WO2003020742A1 (en) * 2001-08-31 2003-03-13 Gen-Probe Incorporated Assay for detection of human parvovirus b19 nucleic acid
US6852491B2 (en) 2001-09-04 2005-02-08 Abbott Laboratories Amplification and detection reagents for HIV-1
US20040054162A1 (en) * 2001-10-30 2004-03-18 Hanna Michelle M. Molecular detection systems utilizing reiterative oligonucleotide synthesis
US7045319B2 (en) * 2001-10-30 2006-05-16 Ribomed Biotechnologies, Inc. Molecular detection systems utilizing reiterative oligonucleotide synthesis
GB0130268D0 (en) * 2001-12-19 2002-02-06 Molecular Light Tech Res Ltd Methods of detecting modification of genetic material and monitoring processes thereof
FR2834521B1 (en) * 2002-01-10 2004-12-17 Bio Merieux METHOD FOR DETECTION AND / OR IDENTIFICATION OF THE ORIGINAL ANIMAL SPECIES OF THE ANIMAL MATERIAL CONTAINED IN A SAMPLE
WO2003064679A2 (en) * 2002-01-30 2003-08-07 Id Biomedical Corporation Methods for detecting vancomycin-resistant microorganisms and compositions therefor
US20030198987A1 (en) * 2002-02-28 2003-10-23 Matveeva Olga V. Methods for designing oligo-probes with high hybridization efficiency and high antisense activity
US7176025B2 (en) * 2002-03-11 2007-02-13 Nugen Technologies, Inc. Methods for generating double stranded DNA comprising a 3′ single stranded portion and uses of these complexes for recombination
US9353405B2 (en) 2002-03-12 2016-05-31 Enzo Life Sciences, Inc. Optimized real time nucleic acid detection processes
US20030219729A1 (en) * 2002-03-15 2003-11-27 Tosoh Corporation Unary avian myeloblastosis virus revers transcriptase and its use
CA2477670A1 (en) * 2002-03-15 2003-09-25 Arcturus Bioscience, Inc. Improved nucleic acid amplification
US7498171B2 (en) 2002-04-12 2009-03-03 Anthrogenesis Corporation Modulation of stem and progenitor cell differentiation, assays, and uses thereof
CA2483694C (en) * 2002-05-17 2016-04-19 Becton, Dickinson And Company Automated system for isolating, amplifying and detecting a target nucleic acid sequence
US20040005614A1 (en) * 2002-05-17 2004-01-08 Nurith Kurn Methods for fragmentation, labeling and immobilization of nucleic acids
PL374195A1 (en) * 2002-06-12 2005-10-03 Chiron Corporation Identification of oligonucleotides for the capture, detection and quantitation of hepatitis a viral nucleic acid
AU2012202286B2 (en) * 2002-06-14 2014-04-03 Gen-Probe Incorporated Compositions and methods for detecting hepatitis B virus
AU2014203075B2 (en) * 2002-06-14 2014-11-27 Gen-Probe Incorporated Compositions and methods for detecting hepatitis B virus
WO2003106714A1 (en) * 2002-06-14 2003-12-24 Gen-Probe Incorporated Compositions and methods for detecting hepatitis b virus
US20040065255A1 (en) * 2002-10-02 2004-04-08 Applied Materials, Inc. Cyclical layer deposition system
US20040259105A1 (en) * 2002-10-03 2004-12-23 Jian-Bing Fan Multiplex nucleic acid analysis using archived or fixed samples
US7115374B2 (en) 2002-10-16 2006-10-03 Gen-Probe Incorporated Compositions and methods for detecting West Nile virus
US7927840B2 (en) 2006-09-11 2011-04-19 Gen Probe Incorporated Method for detecting West Nile Virus nucleic acids in the 3′ non-coding region
US20040101844A1 (en) * 2002-11-21 2004-05-27 Amorese Douglas A. Methods and compositions for producing linearly amplified amounts of (+) strand RNA
WO2004048594A2 (en) 2002-11-21 2004-06-10 Epicentre Technologies Preparation and use of single-stranded transcription substrates for synthesis of transcription products corresponding to target sequences
EP2151507B1 (en) 2002-12-20 2013-02-13 Celera Corporation Genetic polymorphisms associated with myocardial infarction, methods of detection and uses thereof
US6852494B2 (en) * 2003-01-10 2005-02-08 Linden Technologies, Inc. Nucleic acid amplification
WO2004083806A2 (en) 2003-01-22 2004-09-30 University Of South Florida Autonomous genosensor apparatus and methods for use
US20050282170A1 (en) 2003-02-07 2005-12-22 Diagnocure Inc. Method to detect prostate cancer in a sample
US6943768B2 (en) 2003-02-21 2005-09-13 Xtellus Inc. Thermal control system for liquid crystal cell
US20060078893A1 (en) 2004-10-12 2006-04-13 Medical Research Council Compartmentalised combinatorial chemistry by microfluidic control
GB0307403D0 (en) 2003-03-31 2003-05-07 Medical Res Council Selection by compartmentalised screening
GB0307428D0 (en) 2003-03-31 2003-05-07 Medical Res Council Compartmentalised combinatorial chemistry
WO2004092418A2 (en) 2003-04-14 2004-10-28 Nugen Technologies, Inc. Global amplification using a randomly primed composite primer
EP1633893B1 (en) * 2003-05-19 2012-01-25 Gen-Probe Incorporated Compositions, methods and kits for determining the presence of trichomonas vaginalis in a test sample
FR2855832B1 (en) * 2003-06-03 2007-09-14 Biomerieux Sa DIAGNOSTIC AND / OR PROGNOSTIC METHOD OF SEPTIC SYNDROME
FR2857375B1 (en) * 2003-07-10 2007-11-09 Biomerieux Sa PROCESS FOR THE DETECTION AND / OR IDENTIFICATION OF STAPHYLOCOCCUS GENE BACTERIA
ATE544852T1 (en) 2003-07-29 2012-02-15 Otsuka Pharma Co Ltd METHOD FOR ASSESSING THE RISK OF DRUG-INDUCED GRANULOCYTOPENIA
FR2860518B1 (en) * 2003-10-01 2006-02-17 Biomerieux Sa METHOD FOR THE DIAGNOSIS / PROGNOSIS OF NEUROBLASTOMA
KR20070012779A (en) * 2003-10-29 2007-01-29 리보메드 바이오테그놀로지스 인코포레이티드 Compositions, methods and detection technologies for reiterative oligonucleotide synthesis
US7287115B2 (en) * 2003-10-30 2007-10-23 Kabushiki Kaisha Toshiba Multi-chip package type memory system
EP1697535A2 (en) * 2003-11-14 2006-09-06 University of Utah Research Foundation Methods, articles, and compositions for identifying oligonucleotides
CA2547072C (en) 2003-11-26 2015-06-23 Applera Corporation Single nucleotide polymorphisms associated with cardiovascular disorders and statin response, methods of detection and uses thereof
FR2863275B1 (en) * 2003-12-09 2007-08-10 Biomerieux Sa METHOD FOR THE DIAGNOSIS / PROGNOSIS OF BREAST CANCER
DE602004028862D1 (en) * 2003-12-19 2010-10-07 Gen Probe Inc THE NUCLEIC ACIDS OF HIV-1 AND HIV-2
JP2007524407A (en) 2003-12-29 2007-08-30 ニューゲン テクノロジーズ, インコーポレイテッド Methods for analyzing the methylation status of nucleic acids and methods for fragmentation, labeling and immobilization of nucleic acids
ATE481507T1 (en) * 2004-01-23 2010-10-15 Bio Merieux Inc PRIMER AND PROBE DESIGN FOR EFFICIENT AMPLIFICATION AND DETECTION OF THE 3' NON-TRANSLATED AREA OF HCV
FR2866349B1 (en) * 2004-02-12 2008-05-16 Biomerieux Sa METHOD FOR DIAGNOSING / PROGNOSING THROMBOSIS
WO2005087951A2 (en) 2004-03-05 2005-09-22 Gen-Probe Incorporated Reagents, methods and kits for use in deactivating nucleic acids
EP1574583A1 (en) * 2004-03-10 2005-09-14 Roche Diagnostics GmbH Methods for isolation of bacteria from biological samples
FR2868071B1 (en) * 2004-03-26 2006-06-09 Biomerieux Sa MARKING REAGENTS, METHODS FOR SYNTHESIZING SUCH REAGENTS AND METHODS FOR DETECTING BIOLOGICAL MOLECULES
US20050221339A1 (en) 2004-03-31 2005-10-06 Medical Research Council Harvard University Compartmentalised screening by microfluidic control
FR2868433B1 (en) * 2004-04-06 2008-01-18 Biomerieux Sa METHOD FOR THE PROGNOSIS AND / OR DIAGNOSIS OF CANCER
WO2005111241A2 (en) 2004-05-07 2005-11-24 Applera Corporation Genetic polymorphisms associated with liver fibrosis methods of detection and uses thereof
US10066268B2 (en) 2004-05-07 2018-09-04 The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. Methods of diagnosing or treating prostate cancer using the ERG gene, alone or in combination with other over or under expressed genes in prostate cancer
US7311794B2 (en) 2004-05-28 2007-12-25 Wafergen, Inc. Methods of sealing micro wells
US20050277121A1 (en) * 2004-06-11 2005-12-15 Ambion, Inc. Crude biological derivatives competent for nucleic acid detection
EP1781810A1 (en) * 2004-06-29 2007-05-09 Wallac Oy Integrated non-homogeneous nucleic acid amplification and detection
US7776530B2 (en) * 2004-06-29 2010-08-17 Wallac Oy Integrated nucleic acid analysis
EP1767655A4 (en) 2004-07-13 2007-08-01 Takeda Pharmaceutical Method of controlling cell functions
EP2402465B1 (en) 2004-07-13 2014-11-19 Gen-Probe Incorporated Method and kit for detection of Hepatitis A virus nucleic acid
FR2873388B1 (en) * 2004-07-23 2012-04-06 Biomerieux Sa PROCESS FOR MARKING AND PURIFYING NUCLEIC ACIDS OF INTEREST PRESENT IN A BIOLOGICAL SAMPLE TO BE PROCESSED IN A SINGLE REACTION RECIPIENT
US7713697B2 (en) * 2004-08-27 2010-05-11 Gen-Probe Incorporated Methods and kits for amplifying DNA
AU2005280162B2 (en) 2004-08-27 2012-04-26 Gen-Probe Incorporated Single-primer nucleic acid amplification methods
JP5058804B2 (en) 2004-09-14 2012-10-24 アルゴス セラピューティクス,インコーポレイティド Strain-independent amplification of pathogens and vaccines against them
DK2975139T3 (en) 2004-09-30 2019-12-09 Gen Probe Inc ASSAY TO DETECT AND QUANTIFY HIV-1
US7968287B2 (en) 2004-10-08 2011-06-28 Medical Research Council Harvard University In vitro evolution in microfluidic systems
US7354419B2 (en) * 2004-10-15 2008-04-08 Futuremed Interventional, Inc. Medical balloon having strengthening rods
FR2876705B1 (en) * 2004-10-19 2008-12-12 Biomerieux Sa METHOD FOR THE DIAGNOSIS OF ASPIRIN INTOLERANCE
KR20070073935A (en) 2004-10-27 2007-07-10 세페이드 Closed-system multi-stage nucleic acid amplification reactions
CA2585244C (en) 2004-10-28 2015-02-17 Otsuka Pharmaceutical Co., Ltd. Identification marker responsive to interferon therapy for renal cell cancer
CN103146808A (en) 2004-11-03 2013-06-12 意力速分子诊断股份有限公司 Homogeneous analyte detection
US20060223080A1 (en) * 2004-11-09 2006-10-05 Gen-Probe Incorporated Compositions and methods for detecting group a streptococci
CA2491067A1 (en) 2004-12-24 2006-06-24 Stichting Katholieke Universiteit Mrna rations in urinary sediments and/or urine as a prognostic marker for prostate cancer
EP1841879A4 (en) * 2005-01-25 2009-05-27 Population Genetics Technologi Isothermal dna amplification
FR2881437B1 (en) 2005-01-31 2010-11-19 Biomerieux Sa METHOD FOR THE DIAGNOSIS / PROGNOSIS OF A SEPTIC SYNDROME
CA2592179C (en) * 2005-02-07 2012-04-03 Gen-Probe Incorporated Compositions and methods for detecting group b streptococci
US7393665B2 (en) 2005-02-10 2008-07-01 Population Genetics Technologies Ltd Methods and compositions for tagging and identifying polynucleotides
WO2006089154A1 (en) 2005-02-18 2006-08-24 Gen-Probe Incorporated Sample preparation method incorporating an alkaline shock
CA2599013A1 (en) * 2005-02-28 2006-09-08 Gen-Probe Incorporated Compositions and methods of detecting an analyte by using a nucleic acid hybridization switch probe
EP1909108B1 (en) 2005-03-10 2019-05-29 Gen-Probe Incorporated Systems and methods to perform assays for detecting or quantifiying analytes
EP2348320A3 (en) 2005-03-10 2014-06-25 Gen-Probe Incorporated Systems and methods for detecting multiple optical signals
EP1856296A2 (en) 2005-03-11 2007-11-21 Applera Corporation Genetic polymorphisms associated with coronary heart disease, methods of detection and uses thereof
ES2381279T3 (en) 2005-05-06 2012-05-24 Gen-Probe Incorporated Methods and products for capturing target nucleic acid
US8124335B2 (en) 2005-05-06 2012-02-28 Gen-Probe Incorporated Compositions and assays to detect influenza virus A and B nucleic acids
US20060264783A1 (en) 2005-05-09 2006-11-23 Holmes Elizabeth A Systems and methods for monitoring pharmacological parameters
EP1882036B1 (en) 2005-05-17 2012-02-15 Ozgene Pty Ltd Sequential cloning system
FR2886735B1 (en) * 2005-06-01 2015-09-11 Biomerieux Sa PROCESS FOR MARKING OR PROCESSING A BIOLOGICAL SAMPLE CONTAINING BIOLOGICAL MOLECULES OF INTEREST, IN PARTICULAR NUCLEIC ACIDS
WO2006133385A2 (en) 2005-06-06 2006-12-14 Gen-Probe Incorporated Compositions, methods and kits for determining the presence of chlamydophila pneumoniae in a test sample
WO2006131892A2 (en) * 2005-06-09 2006-12-14 Koninklijke Philips Electronics N.V. Amplification of nucleic acids with magnetic detection
US7550264B2 (en) * 2005-06-10 2009-06-23 Datascope Investment Corporation Methods and kits for sense RNA synthesis
US7709197B2 (en) 2005-06-15 2010-05-04 Callida Genomics, Inc. Nucleic acid analysis by random mixtures of non-overlapping fragments
GB0517005D0 (en) * 2005-08-19 2005-09-28 Enigma Diagnostics Ltd Analytical method and kit
US20070054301A1 (en) * 2005-09-06 2007-03-08 Gen-Probe Incorporated Methods, compositions and kits for isothermal amplification of nucleic acids
WO2007030759A2 (en) 2005-09-07 2007-03-15 Nugen Technologies, Inc. Improved nucleic acid amplification procedure
US9957569B2 (en) * 2005-09-12 2018-05-01 The Regents Of The University Of Michigan Recurrent gene fusions in prostate cancer
DE06814528T1 (en) 2005-09-12 2012-01-05 The Regent Of The University Of Michigan RECURRING GENUS FOR PROSTATE CANCER
US7608395B2 (en) 2005-09-15 2009-10-27 Baylor Research Institute Systemic lupus erythematosus diagnostic assay
US7799530B2 (en) 2005-09-23 2010-09-21 Celera Corporation Genetic polymorphisms associated with cardiovascular disorders and drug response, methods of detection and uses thereof
CA2624896C (en) 2005-10-07 2017-11-07 Callida Genomics, Inc. Self-assembled single molecule arrays and uses thereof
AU2006304721B2 (en) 2005-10-17 2012-01-19 Gen-Probe Incorporated Compositions and methods to detect Legionella pneumophila nucleic acid
CN101305101A (en) 2005-11-07 2008-11-12 西门子医疗保健诊断公司 Chlamydia trachomatis specific oligonucleotide sequences
US7831417B2 (en) 2005-11-14 2010-11-09 Gen-Probe Incorporated Parametric calibration method
WO2007120208A2 (en) * 2005-11-14 2007-10-25 President And Fellows Of Harvard College Nanogrid rolling circle dna sequencing
FR2902430B1 (en) * 2005-11-25 2012-11-02 Biomerieux Sa OLIGONUCLEOTIDES, USE, DETECTION METHOD AND KIT FOR DIAGNOSING THE PRESENCE OF INFLUENZA VIRUS H5 AND N1 GENES
US7981606B2 (en) * 2005-12-21 2011-07-19 Roche Molecular Systems, Inc. Control for nucleic acid testing
US20100137163A1 (en) 2006-01-11 2010-06-03 Link Darren R Microfluidic Devices and Methods of Use in The Formation and Control of Nanoreactors
KR101321658B1 (en) 2006-01-18 2013-10-23 아고스 쎄라퓨틱스, 인코포레이티드 Systems and methods for processing samples in a closed container, and related devices
WO2007087262A2 (en) * 2006-01-23 2007-08-02 Population Genetics Technologies Ltd. Selective genome amplification
WO2007092538A2 (en) 2006-02-07 2007-08-16 President And Fellows Of Harvard College Methods for making nucleotide probes for sequencing and synthesis
US20090176878A1 (en) * 2007-10-05 2009-07-09 Washington University In St. Louis Genetic polymorphisms and substance dependence
US20110143344A1 (en) * 2006-03-01 2011-06-16 The Washington University Genetic polymorphisms and substance dependence
RU2394915C2 (en) * 2006-03-24 2010-07-20 Александр Борисович Четверин Non-contact methods of detecting molecular colonies, sets of reagents and device for realising said methods
US8741230B2 (en) 2006-03-24 2014-06-03 Theranos, Inc. Systems and methods of sample processing and fluid control in a fluidic system
US11287421B2 (en) 2006-03-24 2022-03-29 Labrador Diagnostics Llc Systems and methods of sample processing and fluid control in a fluidic system
KR20090028501A (en) 2006-04-07 2009-03-18 지멘스 헬쓰케어 다이아그노스틱스 인크. Neisseria gonorrhoeae specific oligonucleotide sequences
US8900828B2 (en) 2006-05-01 2014-12-02 Cepheid Methods and apparatus for sequential amplification reactions
WO2007133710A2 (en) 2006-05-11 2007-11-22 Raindance Technologies, Inc. Microfluidic devices and methods of use thereof
US9562837B2 (en) 2006-05-11 2017-02-07 Raindance Technologies, Inc. Systems for handling microfludic droplets
AU2007249286B2 (en) * 2006-05-12 2013-06-13 Gen-Probe Incorporated Compositions and methods to detect enterococci nucleic acid
US11001881B2 (en) 2006-08-24 2021-05-11 California Institute Of Technology Methods for detecting analytes
DK2017356T3 (en) 2006-06-06 2012-01-30 Gen Probe Inc Labeled oligonucleotides and their use in methods for amplifying nucleic acids
US8119352B2 (en) * 2006-06-20 2012-02-21 Cepheld Multi-stage amplification reactions by control of sequence replication times
EP2038429B1 (en) * 2006-06-30 2013-08-21 Nugen Technologies, Inc. Methods for fragmentation and labeling of nucleic acids
EP2041574B1 (en) 2006-07-14 2016-11-16 The Regents of The University of California Cancer biomarkers and methods of use threof
WO2008014485A2 (en) 2006-07-28 2008-01-31 California Institute Of Technology Multiplex q-pcr arrays
US11525156B2 (en) 2006-07-28 2022-12-13 California Institute Of Technology Multiplex Q-PCR arrays
JP5635772B2 (en) 2006-08-01 2014-12-03 ジェン−プロウブ インコーポレイテッド Method for capturing non-specific target of nucleic acid
WO2008021123A1 (en) 2006-08-07 2008-02-21 President And Fellows Of Harvard College Fluorocarbon emulsion stabilizing surfactants
WO2008021290A2 (en) 2006-08-09 2008-02-21 Homestead Clinical Corporation Organ-specific proteins and methods of their use
US11560588B2 (en) 2006-08-24 2023-01-24 California Institute Of Technology Multiplex Q-PCR arrays
WO2008030605A2 (en) * 2006-09-08 2008-03-13 The Regents Of The University Of Michigan Herv group ii viruses in lymphoma and cancer
FR2906537A1 (en) 2006-09-28 2008-04-04 Biomerieux Sa METHOD FOR IN VITRO DIAGNOSIS OF BRONCHO-PULMONARY CANCER BY DETECTION OF ALTERNATIVE MAJORITY TRANSCRIPTS OF KLK8 GENE ENCODING KALLICREIN 8 AND USE THEREOF FOR THE PROGNOSIS OF SURVIVAL
WO2008051511A2 (en) 2006-10-20 2008-05-02 Applera Corporation Genetic polymorphisms associated with venous thrombosis, methods of detection and uses thereof
EP1914317A1 (en) * 2006-10-21 2008-04-23 Biolytix AG A method for qualitative and quantitative detection of short nucleic acid sequences of about 8 to 50 nucleotides in length
WO2008058018A2 (en) 2006-11-02 2008-05-15 Mayo Foundation For Medical Education And Research Predicting cancer outcome
EP1921156A1 (en) * 2006-11-10 2008-05-14 bioMerieux B.V. Improved multiplex nucleic acid amplification using blocked primers
US8198027B2 (en) 2006-12-21 2012-06-12 Gen-Probe Incorporated Methods and compositions for nucleic acid amplification
CA2673772A1 (en) * 2007-01-17 2008-07-24 Meridian Bioscience, Inc. Stable reagents and kits useful in loop-mediated isothermal amplification (lamp)
GB0701253D0 (en) 2007-01-23 2007-02-28 Diagnostics For The Real World Nucleic acid amplification and testing
CA2674907C (en) 2007-01-31 2017-04-04 Celera Corporation A molecular prognostic signature for predicting breast cancer distant metastasis, and uses thereof
BRPI0807826A2 (en) 2007-02-02 2014-08-05 Genera Biosystems Ltd "GENERATION OF NUCLEIC ACID MOLECULES".
WO2008097559A2 (en) 2007-02-06 2008-08-14 Brandeis University Manipulation of fluids and reactions in microfluidic systems
US8183359B2 (en) * 2007-03-01 2012-05-22 Gen-Probe Incorporated Kits for amplifying DNA
EP1978111B1 (en) 2007-04-02 2013-03-27 Gen-Probe Incorporated Compositions, kits and related methods for the detection and/or monitoring of Pseudomonas aeruginosa
US8592221B2 (en) 2007-04-19 2013-11-26 Brandeis University Manipulation of fluids, fluid components and reactions in microfluidic systems
WO2008151004A1 (en) 2007-05-31 2008-12-11 Yale University A genetic lesion associated with cancer
FR2917090B1 (en) * 2007-06-11 2012-06-15 Biomerieux Sa MARKING REAGENTS HAVING DIAZO AND NITRO FUNCTIONS, METHODS FOR SYNTHESIZING SUCH REAGENTS AND METHODS FOR DETECTING BIOLOGICAL MOLECULES
CN103418295B (en) 2007-06-21 2015-11-18 简.探针公司 For the instruments and methods of the content of hybrid detection chamber
EP2395113A1 (en) 2007-06-29 2011-12-14 Population Genetics Technologies Ltd. Methods and compositions for isolating nucleic acid sequence variants
CA2707436C (en) * 2007-06-29 2014-01-28 Epicentre Technologies Corporation Copy dna and sense rna
US9303291B2 (en) * 2007-07-06 2016-04-05 The Regents Of The University Of Michigan MIPOL1-ETV1 gene rearrangements
CA2692441C (en) 2007-07-06 2020-01-21 The Regents Of The University Of Michigan Solute carrier family 45 member 3 (slc45a3) and ets family gene fusions in prostate cancer
WO2009012387A2 (en) * 2007-07-18 2009-01-22 Gen-Probe Incorporated Compositions and methods to detect tmprss2/erg transcript variants in prostate cancer
CA2695264C (en) * 2007-08-01 2015-01-27 Dana-Farber Cancer Institute Enrichment of a target sequence
EP2700723B1 (en) 2007-08-06 2015-07-15 Orion Genomics, LLC Novel single nucleotide polymorphisms and combinations of novel and known polymorphisms for determining the allele-specific expression of the IGF2 gene
JP2009077712A (en) 2007-09-11 2009-04-16 F Hoffmann La Roche Ag DIAGNOSTIC TEST FOR SUSCEPTIBILITY TO B-Raf KINASE INHIBITOR
FR2921064A1 (en) * 2007-09-14 2009-03-20 Biomerieux Sa OLIGONUCLEOTIDES, USE, DETECTION METHOD AND KIT FOR DIAGNOSING THE PRESENCE OF THE CHIKUNGUNYA VIRUS E1 GENE
CN101874205B (en) 2007-10-02 2014-10-01 赛拉诺斯股份有限公司 Modular point-of-care devices and uses thereof
CN101910399B (en) 2007-10-30 2015-11-25 考利达基因组股份有限公司 For the device of high throughput sequencing of nucleic acids
WO2009059321A2 (en) 2007-11-01 2009-05-07 University Of Iowa Research Foundation Rca locus analysis to assess susceptibility to amd and mpgnii
US8039212B2 (en) 2007-11-05 2011-10-18 Celera Corporation Genetic polymorphisms associated with liver fibrosis, methods of detection and uses thereof
WO2009061640A2 (en) * 2007-11-06 2009-05-14 Siemens Healthcare Diagnostics Inc. Hepatitis b virus (hbv) specific oligonucleotide sequences
US9551026B2 (en) 2007-12-03 2017-01-24 Complete Genomincs, Inc. Method for nucleic acid detection using voltage enhancement
EP2071034A1 (en) 2007-12-12 2009-06-17 bioMérieux Method for treating a solution in order to destroy any ribonucleic acid after amplification
EP2657351B1 (en) 2007-12-21 2018-06-20 Biomerieux Sa Detection of methicillin-resistant Staphylococcus aureus
US7595164B2 (en) * 2007-12-26 2009-09-29 Gen-Probe Incorporated Compositions and methods to detect Candida albicans nucleic acid
US8034568B2 (en) * 2008-02-12 2011-10-11 Nugen Technologies, Inc. Isothermal nucleic acid amplification methods and compositions
US20090221620A1 (en) 2008-02-20 2009-09-03 Celera Corporation Gentic polymorphisms associated with stroke, methods of detection and uses thereof
GB2470672B (en) 2008-03-21 2012-09-12 Nugen Technologies Inc Methods of RNA amplification in the presence of DNA
US20090304714A1 (en) * 2008-03-25 2009-12-10 The Regents Of The University Of Michigan IKKi Inhibitor Therapies and Screening Methods, and Related IKKi Diagnostics
CA2721536A1 (en) 2008-04-21 2009-10-29 Gen-Probe Incorporated Method for detecting chikungunya virus
WO2009140374A2 (en) 2008-05-13 2009-11-19 Gen-Probe Incorporated Inactivatable target capture oligomers for use in the selective hybridization and capture of target nucleic acid sequences
US8242243B2 (en) 2008-05-15 2012-08-14 Ribomed Biotechnologies, Inc. Methods and reagents for detecting CpG methylation with a methyl CpG binding protein (MBP)
US20110136683A1 (en) 2008-05-28 2011-06-09 Genomedx Biosciences, Inc. Systems and Methods for Expression-Based Discrimination of Distinct Clinical Disease States in Prostate Cancer
JP2011521649A (en) 2008-05-30 2011-07-28 イェール ユニバーシティー Targeted oligonucleotide compositions for modifying gene expression
JP2011521651A (en) * 2008-05-30 2011-07-28 ジェン−プロウブ インコーポレイテッド Compositions, kits and related methods for detection and / or monitoring of Salmonella
US10407731B2 (en) 2008-05-30 2019-09-10 Mayo Foundation For Medical Education And Research Biomarker panels for predicting prostate cancer outcomes
US8216786B2 (en) 2008-07-09 2012-07-10 Celera Corporation Genetic polymorphisms associated with cardiovascular diseases, methods of detection and uses thereof
US8211644B2 (en) * 2008-07-13 2012-07-03 Ribomed Biotechnologies, Inc. Molecular beacon-based methods for detection of targets using abscription
EP4047367A1 (en) 2008-07-18 2022-08-24 Bio-Rad Laboratories, Inc. Method for detecting target analytes with droplet libraries
US20110177509A1 (en) * 2008-07-23 2011-07-21 The Washington University Risk factors and a therapeutic target for neurodegenerative disorders
FR2934595B1 (en) * 2008-07-29 2013-04-05 Biomerieux Sa MARKING REAGENTS HAVING A PYRIDINE CORE HAVING DIAZOMETHYL FUNCTION, METHODS FOR SYNTHESIZING SUCH REAGENTS AND METHODS FOR DETECTING BIOLOGICAL MOLECULES
GB0814570D0 (en) 2008-08-08 2008-09-17 Diagnostics For The Real World Isolation of nucleic acid
WO2010028014A1 (en) * 2008-09-03 2010-03-11 Abbott Laboratories Assays and kits for determining hiv-1 tropism
WO2010030461A2 (en) * 2008-09-12 2010-03-18 Promega Corporation Assessing expression of endogenous and exogenous genes
EP2172563A1 (en) 2008-09-24 2010-04-07 bioMérieux S.A. Method for lowering the dependency towards sequence variation of a nucleic acid target in a diagnostic hybridization assay
US9090948B2 (en) * 2008-09-30 2015-07-28 Abbott Molecular Inc. Primers and probes for detecting human papillomavirus and human beta globin sequences in test samples
US20100301398A1 (en) 2009-05-29 2010-12-02 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes
US8628927B2 (en) 2008-11-07 2014-01-14 Sequenta, Inc. Monitoring health and disease status using clonotype profiles
US9528160B2 (en) 2008-11-07 2016-12-27 Adaptive Biotechnolgies Corp. Rare clonotypes and uses thereof
US9506119B2 (en) 2008-11-07 2016-11-29 Adaptive Biotechnologies Corp. Method of sequence determination using sequence tags
US8748103B2 (en) 2008-11-07 2014-06-10 Sequenta, Inc. Monitoring health and disease status using clonotype profiles
US9394567B2 (en) 2008-11-07 2016-07-19 Adaptive Biotechnologies Corporation Detection and quantification of sample contamination in immune repertoire analysis
US9365901B2 (en) 2008-11-07 2016-06-14 Adaptive Biotechnologies Corp. Monitoring immunoglobulin heavy chain evolution in B-cell acute lymphoblastic leukemia
EP3699296A1 (en) 2008-11-07 2020-08-26 Adaptive Biotechnologies Corporation Methods of monitoring conditions by sequence analysis
US9495515B1 (en) 2009-12-09 2016-11-15 Veracyte, Inc. Algorithms for disease diagnostics
US10236078B2 (en) 2008-11-17 2019-03-19 Veracyte, Inc. Methods for processing or analyzing a sample of thyroid tissue
EP2389449B1 (en) 2008-12-30 2015-02-18 Gen-Probe Incorporated Compositions, kits and related methods for the detection and/or monitoring of listeria
FR2940805B1 (en) 2009-01-05 2015-10-16 Biomerieux Sa METHOD FOR AMPLIFYING AND / OR DETECTING NUCLEIC ACIDS, KITS AND USES THEREOF
CN102639709A (en) 2009-01-09 2012-08-15 密歇根大学董事会 Recurrent gene fusions in cancer
DK3059337T3 (en) 2009-01-15 2019-07-22 Adaptive Biotechnologies Corp Adaptive immunity profiling and methods for producing monoclonal antibodies
CN103923980B (en) 2009-01-19 2017-01-11 生物梅里埃公司 Methods for determining the likelihood of a patient contracting a nosocomial infection and for determining the prognosis of the course of a septic syndrome
JP5586631B2 (en) 2009-01-30 2014-09-10 ジェン−プローブ・インコーポレーテッド System and method for detecting a signal and applying thermal energy to a signal transmission element
US20100286926A1 (en) 2009-02-11 2010-11-11 Orion Genomics Llc Combinations of polymorphisms for determining allele-specific expression of igf2
AU2010217928B2 (en) 2009-02-26 2013-06-06 Gen-Probe Incorporated Assay for detection of human parvovirus nucleic acid
US9074258B2 (en) 2009-03-04 2015-07-07 Genomedx Biosciences Inc. Compositions and methods for classifying thyroid nodule disease
KR20120007002A (en) 2009-03-15 2012-01-19 리보메드 바이오테크놀로지스, 인코퍼레이티드 Abscription based molecular detection
EP3415235A1 (en) 2009-03-23 2018-12-19 Raindance Technologies Inc. Manipulation of microfluidic droplets
WO2010114842A1 (en) 2009-03-30 2010-10-07 Ibis Biosciences, Inc. Bioagent detection systems, devices, and methods
WO2010126913A1 (en) 2009-04-27 2010-11-04 Gen-Probe Incorporated Methods and kits for use in the selective amplification of target sequences
JP6078339B2 (en) 2009-05-07 2017-02-08 ベラサイト インコーポレイテッド Methods and compositions for diagnosis of thyroid status
NZ596296A (en) 2009-05-08 2013-08-30 Novartis Ag Generic assays for detection of influenza viruses
US20120122161A1 (en) 2009-05-22 2012-05-17 Esther Musgrave-Brown Sorting Asymmetrically Tagged Nucleic Acids by Selective Primer Extension
US8776573B2 (en) 2009-05-29 2014-07-15 Life Technologies Corporation Methods and apparatus for measuring analytes
WO2010138187A1 (en) 2009-05-29 2010-12-02 Ion Torrent Systems Incorporated Scaffolded nucleic acid polymer particles and methods of making and using
US20120261274A1 (en) 2009-05-29 2012-10-18 Life Technologies Corporation Methods and apparatus for measuring analytes
CA2768768C (en) 2009-06-23 2021-08-24 Gen-Probe Incorporated Compositions and methods for detecting nucleic acid from mollicutes
AU2010265889A1 (en) 2009-06-25 2012-01-19 Yale University Single nucleotide polymorphisms in BRCA1 and cancer risk
AU2010263172B2 (en) 2009-06-25 2016-03-31 Fred Hutchinson Cancer Research Center Method of measuring adaptive immunity
AU2010266234B2 (en) 2009-07-01 2013-07-25 Gen-Probe Incorporated Methods and compositions for nucleic acid amplification
EP2456887B1 (en) 2009-07-21 2015-11-25 Gen-Probe Incorporated Methods and compositions for quantitative detection of nucleic acid sequences over an extended dynamic range
WO2011021102A2 (en) 2009-08-20 2011-02-24 Population Genetics Technologies Ltd Compositions and methods for intramolecular nucleic acid rearrangement
US20110059453A1 (en) * 2009-08-23 2011-03-10 Affymetrix, Inc. Poly(A) Tail Length Measurement by PCR
JP6057460B2 (en) 2009-08-31 2017-01-11 ジェン−プローブ・インコーポレーテッド Dengue virus assay
CN102712953A (en) * 2009-09-17 2012-10-03 密歇根大学董事会 Recurrent gene fusions in prostate cancer
FR2950358B1 (en) 2009-09-18 2015-09-11 Biomerieux Sa SIMPLIFIED NUCLEIC ACID AMPLIFICATION DEVICE AND METHOD FOR IMPLEMENTING THE SAME
US10520500B2 (en) 2009-10-09 2019-12-31 Abdeslam El Harrak Labelled silica-based nanomaterial with enhanced properties and uses thereof
US20120245041A1 (en) 2009-11-04 2012-09-27 Sydney Brenner Base-by-base mutation screening
KR20170098985A (en) 2009-11-19 2017-08-30 솔리스 바이오다인 Compositions for increasing polypeptide stability and activity, and related methods
US9121054B2 (en) * 2009-12-08 2015-09-01 Biohelix Corporation Detection of nucleic acid amplification products in the presence of an internal control sequence on an immunochromatographic strip
US10446272B2 (en) 2009-12-09 2019-10-15 Veracyte, Inc. Methods and compositions for classification of samples
WO2011079176A2 (en) 2009-12-23 2011-06-30 Raindance Technologies, Inc. Microfluidic systems and methods for reducing the exchange of molecules between droplets
US8790879B2 (en) * 2010-01-22 2014-07-29 Gen-Probe Incorporated Probes for detecting the presence of Trichomonas vaginalis in a sample
US20110312503A1 (en) 2010-01-23 2011-12-22 Artemis Health, Inc. Methods of fetal abnormality detection
CN102725424B (en) 2010-01-25 2014-07-09 Rd生物科技公司 Self-folding amplification of target nucleic acid
TWI518325B (en) 2010-02-04 2016-01-21 自治醫科大學 Identification, assessment, and therapy of cancers with innate or acquired resistance to alk inhibitors
EP2516681B1 (en) * 2010-02-11 2017-10-18 Nanostring Technologies, Inc Compositions and methods for the detection of preferably small rnas by bridge hybridisation and ligation
US9366632B2 (en) 2010-02-12 2016-06-14 Raindance Technologies, Inc. Digital analyte analysis
US9399797B2 (en) 2010-02-12 2016-07-26 Raindance Technologies, Inc. Digital analyte analysis
US8535889B2 (en) 2010-02-12 2013-09-17 Raindance Technologies, Inc. Digital analyte analysis
US10351905B2 (en) 2010-02-12 2019-07-16 Bio-Rad Laboratories, Inc. Digital analyte analysis
EP3040425B1 (en) 2010-02-17 2019-08-28 Gen-Probe Incorporated Compostions and methods to detect atopobium vaginae nucleic acid
US20130053253A1 (en) 2010-02-22 2013-02-28 Population Genetics Technologies Ltd Region of Interest Extraction and Normalization Methods
US10113206B2 (en) 2010-02-24 2018-10-30 Grifols Therapeutics Inc. Methods, compositions, and kits for determining human immunodeficiency virus (HIV)
WO2011107887A2 (en) 2010-03-02 2011-09-09 Population Genetic Technologies Ltd. Methods for replicating polynucleotides with secondary structure
US8623603B2 (en) 2010-03-08 2014-01-07 Dana-Farber Cancer Institute, Inc. Full cold-PCR enrichment with reference blocking sequence
WO2011128096A1 (en) 2010-04-16 2011-10-20 Roche Diagnostics Gmbh Polymorphism markers for predicting response to interleukin-6 receptor-inhibiting monoclonal antibody drug treatment
US20110269735A1 (en) 2010-04-19 2011-11-03 Celera Corporation Genetic polymorphisms associated with statin response and cardiovascular diseases, methods of detection and uses thereof
CA3011697C (en) 2010-04-21 2020-04-07 Gen-Probe Incorporated Compositions, methods and kits to detect herpes simplex virus nucleic acids
WO2011139714A2 (en) 2010-04-26 2011-11-10 Atyr Pharma, Inc. Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of cysteinyl-trna synthetase
EP2564200B8 (en) 2010-04-27 2019-10-02 The Regents of The University of California Cancer biomarkers and methods of use thereof
US8961960B2 (en) 2010-04-27 2015-02-24 Atyr Pharma, Inc. Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of isoleucyl tRNA synthetases
CN103097524B (en) 2010-04-28 2016-08-03 Atyr医药公司 The innovation for the treatment of, diagnosis and the antibody compositions relevant to the protein fragments of Alanyl-tRNA synthetase finds
US9068177B2 (en) 2010-04-29 2015-06-30 Atyr Pharma, Inc Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of glutaminyl-tRNA synthetases
EP2563912B1 (en) 2010-04-29 2018-09-05 aTyr Pharma, Inc. Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of asparaginyl trna synthetases
AU2011248457B2 (en) 2010-04-29 2017-02-16 Pangu Biopharma Limited Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of valyl tRNA synthetases
US8774494B2 (en) 2010-04-30 2014-07-08 Complete Genomics, Inc. Method and system for accurate alignment and registration of array for DNA sequencing
JP6008840B2 (en) 2010-05-03 2016-10-19 エータイアー ファーマ, インコーポレイテッド Innovative discovery of therapeutic, diagnostic and antibody compositions related to protein fragments of phenylalanyl αtRNA synthetase
AU2011248230B2 (en) 2010-05-03 2016-10-06 Pangu Biopharma Limited Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of methionyl-tRNA synthetases
CN103096925A (en) 2010-05-03 2013-05-08 Atyr医药公司 Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of arginyl-tRNA synthetases
AU2011248101B2 (en) 2010-05-04 2016-10-20 Atyr Pharma, Inc. Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of p38 multi-tRNA synthetase complex
CA2798123C (en) 2010-05-05 2020-06-23 The Governing Council Of The University Of Toronto Method of processing dried samples using digital microfluidic device
WO2011138402A1 (en) 2010-05-05 2011-11-10 Check-Points Holding B.V. Assays, compositions and methods for detecting drug resistant micro-organisms
ES2593614T3 (en) 2010-05-06 2016-12-12 Adaptive Biotechnologies Corporation Health and disease status monitoring using clonotype profiles
AU2011252990B2 (en) 2010-05-14 2017-04-20 Pangu Biopharma Limited Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of phenylalanyl-beta-tRNA synthetases
JP5906237B2 (en) 2010-06-01 2016-04-20 エータイアー ファーマ, インコーポレイテッド Innovative discovery of therapeutic, diagnostic and antibody compositions related to protein fragments of lysyl tRNA synthetase
EP2576837B1 (en) 2010-06-04 2017-09-06 Chronix Biomedical Prostate cancer associated circulating nucleic acid biomarkers
JP5916718B2 (en) 2010-06-04 2016-05-11 ビオメリューBiomerieux Method and kit for determining prognosis of colorectal cancer
EP3290529B1 (en) 2010-06-11 2019-05-22 Life Technologies Corporation Alternative nucleotide flows in sequencing-by-synthesis methods
WO2011161549A2 (en) 2010-06-24 2011-12-29 Population Genetics Technologies Ltd. Methods and compositions for polynucleotide library production, immortalization and region of interest extraction
EP2588629B1 (en) 2010-06-30 2017-05-17 Gen-Probe Incorporated Method and apparatus for identifying analyte-containing samples using single-read determination of analyte and process control signals
US8790878B2 (en) 2010-07-07 2014-07-29 The Regents Of The University Of Michigan Diagnosis and treatment of breast cancer
US9234249B2 (en) 2010-07-12 2016-01-12 Gen-Probe Incorporated Compositions and assays to detect swine H1N1 influenza A virus, seasonal H1 influenza A virus and seasonal H3 influenza A virus nucleic acids
EP2593125B1 (en) 2010-07-12 2017-11-01 aTyr Pharma, Inc. Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of glycyl-trna synthetases
CN103540518B (en) 2010-07-23 2015-07-08 贝克曼考尔特公司 Test box
EP3578205A1 (en) 2010-08-06 2019-12-11 ModernaTX, Inc. A pharmaceutical formulation comprising engineered nucleic acids and medical use thereof
US11031095B2 (en) 2010-08-06 2021-06-08 Ariosa Diagnostics, Inc. Assay systems for determination of fetal copy number variation
US10533223B2 (en) 2010-08-06 2020-01-14 Ariosa Diagnostics, Inc. Detection of target nucleic acids using hybridization
US20140342940A1 (en) 2011-01-25 2014-11-20 Ariosa Diagnostics, Inc. Detection of Target Nucleic Acids using Hybridization
US11203786B2 (en) 2010-08-06 2021-12-21 Ariosa Diagnostics, Inc. Detection of target nucleic acids using hybridization
US8700338B2 (en) 2011-01-25 2014-04-15 Ariosa Diagnosis, Inc. Risk calculation for evaluation of fetal aneuploidy
US20120034603A1 (en) 2010-08-06 2012-02-09 Tandem Diagnostics, Inc. Ligation-based detection of genetic variants
US20130040375A1 (en) 2011-08-08 2013-02-14 Tandem Diagnotics, Inc. Assay systems for genetic analysis
US20130261003A1 (en) 2010-08-06 2013-10-03 Ariosa Diagnostics, In. Ligation-based detection of genetic variants
US10167508B2 (en) 2010-08-06 2019-01-01 Ariosa Diagnostics, Inc. Detection of genetic abnormalities
CN103221810B (en) 2010-08-18 2016-08-03 生命科技股份有限公司 Immersion coating for the micropore of electrochemical detection device
JP5964304B2 (en) 2010-08-25 2016-08-03 エータイアー ファーマ, インコーポレイテッド Innovative discovery of therapeutic, diagnostic and antibody compositions related to protein fragments of tyrosyl-tRNA synthetase
WO2012030856A2 (en) 2010-08-30 2012-03-08 Gen-Probe Incorporated Compositions, methods and reaction mixtures for the detection of xenotropic murine leukemia virus-related virus
WO2012032482A1 (en) 2010-09-07 2012-03-15 Novartis Ag Generic assays for detection of mammalian reovirus
EP3327140A1 (en) 2010-09-16 2018-05-30 Gen-Probe Incorporated Capture probes immobilizable via l-nucleotide tail
CN103154273A (en) 2010-09-21 2013-06-12 群体遗传学科技有限公司 Increasing confidence of allele calls with molecular counting
US20140004510A1 (en) 2010-09-24 2014-01-02 Massachusetts Eye And Ear Infirmary Methods and compositions for prognosing and/or detecting age-related macular degeneration
US9110079B2 (en) 2010-09-29 2015-08-18 Biomerieux Method and kit for establishing an in vitro prognosis on a patient exhibiting SIRS
US9562897B2 (en) 2010-09-30 2017-02-07 Raindance Technologies, Inc. Sandwich assays in droplets
ES2925251T3 (en) 2010-10-01 2022-10-14 Modernatx Inc Ribonucleic Acids Containing N1-Methyl-Pseudoracils and Uses Thereof
EP2625297B1 (en) 2010-10-04 2018-10-10 Gen-Probe Prodesse, Inc. Compositions, methods and kits to detect adenovirus nucleic acids
US8725422B2 (en) 2010-10-13 2014-05-13 Complete Genomics, Inc. Methods for estimating genome-wide copy number variations
US10233501B2 (en) 2010-10-19 2019-03-19 Northwestern University Biomarkers predictive of predisposition to depression and response to treatment
US20150218639A1 (en) 2014-01-17 2015-08-06 Northwestern University Biomarkers predictive of predisposition to depression and response to treatment
US20150225792A1 (en) 2014-01-17 2015-08-13 Northwestern University Compositions and methods for identifying depressive disorders
US10093981B2 (en) 2010-10-19 2018-10-09 Northwestern University Compositions and methods for identifying depressive disorders
US8563298B2 (en) 2010-10-22 2013-10-22 T2 Biosystems, Inc. NMR systems and methods for the rapid detection of analytes
RU2653451C2 (en) 2010-10-22 2018-05-08 Т2 Байосистемз, Инк. System (variants) and method of detecting analyte presence in liquid sample
US20120108651A1 (en) 2010-11-02 2012-05-03 Leiden University Medical Center (LUMC) Acting on Behalf of Academic Hospital Leiden (AZL) Genetic polymorphisms associated with venous thrombosis and statin response, methods of detection and uses thereof
EP3336200A1 (en) 2010-11-19 2018-06-20 The Regents Of The University Of Michigan Prostate cancer ncrna and uses thereof
US8945556B2 (en) 2010-11-19 2015-02-03 The Regents Of The University Of Michigan RAF gene fusions
US9594870B2 (en) 2010-12-29 2017-03-14 Life Technologies Corporation Time-warped background signal for sequencing-by-synthesis operations
WO2013025998A1 (en) 2011-08-18 2013-02-21 Life Technologies Corporation Methods, systems, and computer readable media for making base calls in nucleic acid sequencing
EP2658999B1 (en) 2010-12-30 2019-03-13 Life Technologies Corporation Models for analyzing data from sequencing-by-synthesis operations
US10241075B2 (en) 2010-12-30 2019-03-26 Life Technologies Corporation Methods, systems, and computer readable media for nucleic acid sequencing
US20130060482A1 (en) 2010-12-30 2013-03-07 Life Technologies Corporation Methods, systems, and computer readable media for making base calls in nucleic acid sequencing
CA2825196C (en) 2011-01-21 2021-01-05 Theranos, Inc. Systems and methods for sample use maximization
US9994897B2 (en) 2013-03-08 2018-06-12 Ariosa Diagnostics, Inc. Non-invasive fetal sex determination
WO2012103031A2 (en) 2011-01-25 2012-08-02 Ariosa Diagnostics, Inc. Detection of genetic abnormalities
US8756020B2 (en) 2011-01-25 2014-06-17 Ariosa Diagnostics, Inc. Enhanced risk probabilities using biomolecule estimations
US10131947B2 (en) 2011-01-25 2018-11-20 Ariosa Diagnostics, Inc. Noninvasive detection of fetal aneuploidy in egg donor pregnancies
US11270781B2 (en) 2011-01-25 2022-03-08 Ariosa Diagnostics, Inc. Statistical analysis for non-invasive sex chromosome aneuploidy determination
FR2970975B1 (en) 2011-01-27 2016-11-04 Biomerieux Sa METHOD AND KIT FOR DETERMINING IN VITRO THE IMMUNE STATUS OF AN INDIVIDUAL
US9365897B2 (en) 2011-02-08 2016-06-14 Illumina, Inc. Selective enrichment of nucleic acids
EP3412778A1 (en) 2011-02-11 2018-12-12 Raindance Technologies, Inc. Methods for forming mixed droplets
EP2675275B1 (en) 2011-02-14 2017-12-20 The Regents Of The University Of Michigan Compositions and methods for the treatment of obesity and related disorders
EP3736281A1 (en) 2011-02-18 2020-11-11 Bio-Rad Laboratories, Inc. Compositions and methods for molecular labeling
AU2012222178B2 (en) 2011-02-24 2014-12-18 Gen-Probe Incorporated Systems and methods for distinguishing optical signals of different modulation frequencies in an optical signal detector
KR20140006963A (en) 2011-02-25 2014-01-16 노파르티스 아게 Exogenous internal positive control
US20120219950A1 (en) 2011-02-28 2012-08-30 Arnold Oliphant Assay systems for detection of aneuploidy and sex determination
EP2683833B1 (en) 2011-03-10 2018-09-26 Gen-Probe Incorporated Methods for the selection and optimization of oligonucleotide tag sequences
WO2012129758A1 (en) 2011-03-25 2012-10-04 Biomerieux Method and kit for determining in vitro probability for individual to suffer from colorectal cancer
EP2691101A2 (en) 2011-03-31 2014-02-05 Moderna Therapeutics, Inc. Delivery and formulation of engineered nucleic acids
EP3333269B1 (en) 2011-03-31 2021-05-05 Dana-Farber Cancer Institute, Inc. Methods to enable multiplex cold-pcr
CN105861645B (en) 2011-04-08 2020-02-21 生命科技股份有限公司 Phase-protected reagent flow ordering for use in sequencing-by-synthesis
RU2690374C2 (en) 2011-04-15 2019-06-03 Бектон, Дикинсон Энд Компани Scanning in real time microfluid thermal cycler and methods of synchronized thermal cycling and scanning optical detection
EP2702166B1 (en) 2011-04-25 2018-06-06 Gen-Probe Incorporated Compositions and methods for detecting bv-associated bacterial nucleic acid
WO2012167142A2 (en) 2011-06-02 2012-12-06 Raindance Technolgies, Inc. Enzyme quantification
US8841071B2 (en) 2011-06-02 2014-09-23 Raindance Technologies, Inc. Sample multiplexing
HUE036963T2 (en) 2011-06-15 2018-08-28 Grifols Therapeutics Inc Methods, compositions, and kits for determining human immunodeficiency virus (hiv)
US9752201B2 (en) 2011-07-15 2017-09-05 Gen-Probe Incorporated Compositions and method for detecting human parvovirus nucleic acid and for detecting hepatitis A virus nucleic acids in single-plex or multiplex assays
US8658430B2 (en) 2011-07-20 2014-02-25 Raindance Technologies, Inc. Manipulating droplet size
EP2751280B1 (en) 2011-08-31 2016-06-29 F.Hoffmann-La Roche Ag Method for predicting risk of hypertension associated with anti-angiogenesis therapy
JP2014526900A (en) 2011-08-31 2014-10-09 エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト Responsiveness to angiogenesis inhibitors
US10704164B2 (en) 2011-08-31 2020-07-07 Life Technologies Corporation Methods, systems, computer readable media, and kits for sample identification
EP4219741A3 (en) 2011-09-06 2023-08-23 Gen-Probe Incorporated Closed nucleic acid structures
US10752944B2 (en) 2011-09-06 2020-08-25 Gen-Probe Incorporated Circularized templates for sequencing
US8712697B2 (en) 2011-09-07 2014-04-29 Ariosa Diagnostics, Inc. Determination of copy number variations using binomial probability calculations
BR112013031802A2 (en) 2011-09-07 2016-12-20 Grifols Therapeutics Inc isolated nucleic acid molecule, composition, methods for amplifying a target sequence, and for determining hav in a sample, and kit
WO2013036928A1 (en) 2011-09-08 2013-03-14 Gen-Probe Incorporated Compositions and methods for detecting bv-associated bacterial nucleic acid
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US10385475B2 (en) 2011-09-12 2019-08-20 Adaptive Biotechnologies Corp. Random array sequencing of low-complexity libraries
US8435738B2 (en) 2011-09-25 2013-05-07 Theranos, Inc. Systems and methods for multi-analysis
US9664702B2 (en) 2011-09-25 2017-05-30 Theranos, Inc. Fluid handling apparatus and configurations
US8380541B1 (en) 2011-09-25 2013-02-19 Theranos, Inc. Systems and methods for collecting and transmitting assay results
US20140170735A1 (en) 2011-09-25 2014-06-19 Elizabeth A. Holmes Systems and methods for multi-analysis
US9268915B2 (en) 2011-09-25 2016-02-23 Theranos, Inc. Systems and methods for diagnosis or treatment
US8475739B2 (en) 2011-09-25 2013-07-02 Theranos, Inc. Systems and methods for fluid handling
US8840838B2 (en) 2011-09-25 2014-09-23 Theranos, Inc. Centrifuge configurations
US9632102B2 (en) 2011-09-25 2017-04-25 Theranos, Inc. Systems and methods for multi-purpose analysis
US9619627B2 (en) 2011-09-25 2017-04-11 Theranos, Inc. Systems and methods for collecting and transmitting assay results
CA2846630A1 (en) 2011-09-19 2013-03-28 Genentech, Inc. Combination treatments comprising c-met antagonists and b-raf antagonists
WO2013044097A1 (en) 2011-09-21 2013-03-28 Gen-Probe Incorporated Methods for amplifying nucleic acid using tag-mediated displacement
US9810704B2 (en) 2013-02-18 2017-11-07 Theranos, Inc. Systems and methods for multi-analysis
US9250229B2 (en) 2011-09-25 2016-02-02 Theranos, Inc. Systems and methods for multi-analysis
US10012664B2 (en) 2011-09-25 2018-07-03 Theranos Ip Company, Llc Systems and methods for fluid and component handling
JP2014530358A (en) 2011-09-25 2014-11-17 セラノス, インコーポレイテッド System and method for multiplex analysis
CN110511939A (en) 2011-10-03 2019-11-29 现代泰克斯公司 Nucleosides, nucleotide and nucleic acid of modification and application thereof
CN104080958A (en) 2011-10-19 2014-10-01 纽亘技术公司 Compositions and methods for directional nucleic acid amplification and sequencing
PL2768985T3 (en) 2011-10-21 2019-10-31 Chronix Biomedical Colorectal cancer associated circulating nucleic acid biomarkers
AU2012325791B2 (en) 2011-10-21 2018-04-05 Adaptive Biotechnologies Corporation Quantification of adaptive immune cell genomes in a complex mixture of cells
US10837879B2 (en) 2011-11-02 2020-11-17 Complete Genomics, Inc. Treatment for stabilizing nucleic acid arrays
JP2014533100A (en) 2011-11-04 2014-12-11 オスロ ウニヴェルスィテーツスィーケフース ハーエフOslo Universitetssykehus Hf Methods and biomarkers for the analysis of colorectal cancer
WO2013067413A1 (en) 2011-11-04 2013-05-10 Gen-Probe Incorporated Molecular assay reagents and methods
BR112014010955A2 (en) 2011-11-07 2017-06-06 Beckman Coulter Inc system and method for processing samples
KR20140091033A (en) 2011-11-07 2014-07-18 베크만 컬터, 인코포레이티드 Specimen container detection
KR102040996B1 (en) 2011-11-07 2019-11-05 베크만 컬터, 인코포레이티드 Robotic arm
CN104040357B (en) 2011-11-07 2016-11-23 贝克曼考尔特公司 Halver system and workflow
BR112014011046A2 (en) 2011-11-07 2017-06-13 Beckman Coulter, Inc. workflow and centrifuge system
US8973736B2 (en) 2011-11-07 2015-03-10 Beckman Coulter, Inc. Magnetic damping for specimen transport system
RU2014123166A (en) 2011-11-23 2015-12-27 Ф.Хоффманн-Ля Рош Аг SUSCEPTIBILITY TO ANGIOGENESIS INHIBITORS
DE102011120550B4 (en) 2011-12-05 2013-11-07 Gen-Probe Prodesse, Inc. Compositions, methods and kits for the detection of adenovirus nucleic acids
WO2013086450A1 (en) 2011-12-09 2013-06-13 Adaptive Biotechnologies Corporation Diagnosis of lymphoid malignancies and minimal residual disease detection
US10513737B2 (en) 2011-12-13 2019-12-24 Decipher Biosciences, Inc. Cancer diagnostics using non-coding transcripts
US9499865B2 (en) 2011-12-13 2016-11-22 Adaptive Biotechnologies Corp. Detection and measurement of tissue-infiltrating lymphocytes
CA3018046A1 (en) 2011-12-16 2013-06-20 Moderna Therapeutics, Inc. Modified nucleoside, nucleotide, and nucleic acid compositions
WO2013096460A1 (en) 2011-12-20 2013-06-27 The Regents Of The University Of Michigan Pseudogenes and uses thereof
US9334491B2 (en) 2011-12-22 2016-05-10 Ibis Biosciences, Inc. Systems and methods for isolating nucleic acids from cellular samples
US9803188B2 (en) 2011-12-22 2017-10-31 Ibis Biosciences, Inc. Systems and methods for isolating nucleic acids
US9115394B2 (en) 2011-12-22 2015-08-25 Roche Molecular Systems, Inc. Methods and reagents for reducing non-specific amplification
EP3450572A1 (en) 2011-12-23 2019-03-06 bioMérieux Detection of meca variant strains of methicillin-resistant staphylococcus aureus
WO2013101935A1 (en) 2011-12-27 2013-07-04 Ibis Biosciences, Inc. Bioagent detection oligonucleotides
WO2013101783A2 (en) 2011-12-30 2013-07-04 Bio-Rad Laboratories, Inc. Methods and compositions for performing nucleic acid amplification reactions
US9822417B2 (en) 2012-01-09 2017-11-21 Oslo Universitetssykehus Hf Methods and biomarkers for analysis of colorectal cancer
US9194840B2 (en) 2012-01-19 2015-11-24 Life Technologies Corporation Sensor arrays and methods for making same
GB2533882B (en) 2012-01-26 2016-10-12 Nugen Tech Inc Method of enriching and sequencing nucleic acids of interest using massively parallel sequencing
US9864846B2 (en) 2012-01-31 2018-01-09 Life Technologies Corporation Methods and computer program products for compression of sequencing data
US9515676B2 (en) 2012-01-31 2016-12-06 Life Technologies Corporation Methods and computer program products for compression of sequencing data
EP2814514B1 (en) 2012-02-16 2017-09-13 Atyr Pharma, Inc. Histidyl-trna synthetases for treating autoimmune and inflammatory diseases
WO2013124738A2 (en) 2012-02-21 2013-08-29 Oslo Universitetssykehus Hf Methods and biomarkers for detection and prognosis of cervical cancer
WO2013124743A1 (en) 2012-02-22 2013-08-29 Population Genetics Technologies Ltd. Compositions and methods for intramolecular nucleic acid rearrangement ii
AU2013205010B2 (en) 2012-02-24 2016-10-20 Gen-Probe Incorporated Detection of Shiga toxin genes in bacteria
EP2820155B1 (en) 2012-02-28 2017-07-26 Population Genetics Technologies Ltd. Method for attaching a counter sequence to a nucleic acid sample
EP2823060B1 (en) 2012-03-05 2018-02-14 Adaptive Biotechnologies Corporation Determining paired immune receptor chains from frequency matched subunits
CA2866254A1 (en) 2012-03-06 2013-09-12 Oslo Universitetssykehus Hf Gene signatures associated with efficacy of postmastectomy radiotherapy in breast cancer
JP6270814B2 (en) 2012-03-29 2018-01-31 ベクトン・ディキンソン・アンド・カンパニーBecton, Dickinson And Company Nucleic acids for nucleic acid amplification
US9803239B2 (en) 2012-03-29 2017-10-31 Complete Genomics, Inc. Flow cells for high density array chips
US9572897B2 (en) 2012-04-02 2017-02-21 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9283287B2 (en) 2012-04-02 2016-03-15 Moderna Therapeutics, Inc. Modified polynucleotides for the production of nuclear proteins
US8999380B2 (en) 2012-04-02 2015-04-07 Moderna Therapeutics, Inc. Modified polynucleotides for the production of biologics and proteins associated with human disease
US10501512B2 (en) 2012-04-02 2019-12-10 Modernatx, Inc. Modified polynucleotides
EP2834370B1 (en) 2012-04-03 2019-01-02 The Regents Of The University Of Michigan Biomarker associated with irritable bowel syndrome and crohn's disease
IN2014DN09302A (en) 2012-04-13 2015-07-10 Becton Dickinson Co
SG10201802883UA (en) 2012-04-19 2018-05-30 Life Technologies Corp Nucleic acid amplification
EP2839026B1 (en) 2012-04-19 2016-08-10 Life Technologies Corporation Nucleic acid amplification
AU2013205110B2 (en) 2012-04-24 2016-10-13 Gen-Probe Incorporated Compositions, Methods and Kits to Detect Herpes Simplex Virus Nucleic Acids
HUE029357T2 (en) 2012-05-08 2017-02-28 Adaptive Biotechnologies Corp Compositions and method for measuring and calibrating amplification bias in multiplexed pcr reactions
US9646132B2 (en) 2012-05-11 2017-05-09 Life Technologies Corporation Models for analyzing data from sequencing-by-synthesis operations
US9133490B2 (en) 2012-05-16 2015-09-15 Transgenomic, Inc. Step-up method for COLD-PCR enrichment
US10289800B2 (en) 2012-05-21 2019-05-14 Ariosa Diagnostics, Inc. Processes for calculating phased fetal genomic sequences
EP4148142A1 (en) 2012-05-21 2023-03-15 The Scripps Research Institute Methods of sample preparation
AU2013205064B2 (en) 2012-06-04 2015-07-30 Gen-Probe Incorporated Compositions and Methods for Amplifying and Characterizing HCV Nucleic Acid
US9628676B2 (en) 2012-06-07 2017-04-18 Complete Genomics, Inc. Imaging systems with movable scan mirrors
US9488823B2 (en) 2012-06-07 2016-11-08 Complete Genomics, Inc. Techniques for scanned illumination
AU2013202804A1 (en) 2012-06-14 2014-01-16 Gen-Probe Incorporated Use of a fluorescent material to detect failure or deteriorated performance of a fluorometer
SG11201408478QA (en) 2012-06-18 2015-02-27 Nugen Technologies Inc Compositions and methods for negative selection of non-desired nucleic acid sequences
WO2014005076A2 (en) 2012-06-29 2014-01-03 The Regents Of The University Of Michigan Methods and biomarkers for detection of kidney disorders
US20150011396A1 (en) 2012-07-09 2015-01-08 Benjamin G. Schroeder Methods for creating directional bisulfite-converted nucleic acid libraries for next generation sequencing
AU2013205087B2 (en) 2012-07-13 2016-03-03 Gen-Probe Incorporated Method for detecting a minority genotype
US20140024015A1 (en) 2012-07-18 2014-01-23 Idexx Laboratories, Inc. Boone Cardiovirus
US9206417B2 (en) 2012-07-19 2015-12-08 Ariosa Diagnostics, Inc. Multiplexed sequential ligation-based detection of genetic variants
WO2014018885A2 (en) 2012-07-27 2014-01-30 Gen-Probe Incorporated Dual reference calibration method and system for quantifying polynucleotides
AU2013202808B2 (en) 2012-07-31 2014-11-13 Gen-Probe Incorporated System and method for performing multiplex thermal melt analysis
AU2013202793B2 (en) 2012-07-31 2014-09-18 Gen-Probe Incorporated System, method and apparatus for automated incubation
EP2885640B1 (en) 2012-08-16 2018-07-18 Genomedx Biosciences, Inc. Prostate cancer prognostics using biomarkers
CA2883219C (en) 2012-08-30 2020-12-29 Gen-Probe Incorporated Multiphase nucleic acid amplification
WO2014043143A1 (en) 2012-09-11 2014-03-20 Life Technologies Corporation Nucleic acid amplification
WO2014042986A1 (en) 2012-09-11 2014-03-20 Theranos, Inc. Information management systems and methods using a biological signature
EP2895620B1 (en) 2012-09-11 2017-08-02 Life Technologies Corporation Nucleic acid amplification
US9914968B2 (en) 2012-09-26 2018-03-13 Cepheid Honeycomb tube
JP6449160B2 (en) 2012-10-01 2019-01-09 アダプティブ バイオテクノロジーズ コーポレイション Cross-reference of applications related to immune competence assessment by adaptive immune receptor diversity and clonal characterization
EP2904120B1 (en) 2012-10-04 2018-01-10 The Board of Trustees of The Leland Stanford Junior University Methods and reagents for detection, quantitation, and serotyping of dengue viruses
US10329608B2 (en) 2012-10-10 2019-06-25 Life Technologies Corporation Methods, systems, and computer readable media for repeat sequencing
AU2013205122B2 (en) 2012-10-11 2016-11-10 Gen-Probe Incorporated Compositions and Methods for Detecting Human Papillomavirus Nucleic Acid
EP2909341A2 (en) 2012-10-18 2015-08-26 Oslo Universitetssykehus HF Biomarkers for cervical cancer
WO2015160439A2 (en) 2014-04-17 2015-10-22 Adaptive Biotechnologies Corporation Quantification of adaptive immune cell genomes in a complex mixture of cells
RS63237B1 (en) 2012-11-26 2022-06-30 Modernatx Inc Terminally modified rna
CN104937111B (en) 2012-11-27 2018-05-11 智利天主教教皇大学 For diagnosing the composition and method of thyroid tumors
AU2013205090B2 (en) 2012-12-07 2016-07-28 Gen-Probe Incorporated Compositions and Methods for Detecting Gastrointestinal Pathogen Nucleic Acid
FR3000966B1 (en) 2013-01-11 2016-10-28 Biomerieux Sa METHOD FOR ESTABLISHING IN VITRO A PROGNOSIS OF SEVERITY IN A SEPTIC SHOCK PATIENT
CA2897474A1 (en) 2013-01-24 2014-07-31 California Institute Of Technology Chromophore-based characterization and detection methods
US10077475B2 (en) 2013-01-24 2018-09-18 California Institute Of Technology FRET-based analytes detection and related methods and systems
MX354033B (en) 2013-02-18 2018-02-09 Theranos Ip Co Llc Systems and methods for collecting and transmitting assay results.
US10640808B2 (en) 2013-03-13 2020-05-05 Abbott Molecular Inc. Systems and methods for isolating nucleic acids
EP2971115B1 (en) 2013-03-13 2022-07-27 Seegene, Inc. Quantification of target nucleic acid using melting peak analysis
CA2905429A1 (en) 2013-03-14 2014-10-02 Abbott Molecular Inc. Minimizing errors using uracil-dna-n-glycosylase
US20140296080A1 (en) 2013-03-14 2014-10-02 Life Technologies Corporation Methods, Systems, and Computer Readable Media for Evaluating Variant Likelihood
AU2013202778A1 (en) 2013-03-14 2014-10-02 Gen-Probe Incorporated Systems, methods, and apparatuses for performing automated reagent-based assays
AU2013202805B2 (en) 2013-03-14 2015-07-16 Gen-Probe Incorporated System and method for extending the capabilities of a diagnostic analyzer
AU2013202788B2 (en) 2013-03-14 2015-10-01 Gen-Probe Incorporated Indexing signal detection module
CN105378107A (en) 2013-03-14 2016-03-02 雅培分子公司 Multiplex methylation-specific amplification systems and methods
CA2905144C (en) 2013-03-14 2023-08-22 Lyle J. Arnold Methods for amplification of nucleic acids on solid support
US9976175B2 (en) 2013-03-15 2018-05-22 Gen-Probe Incorporated Calibration method, apparatus and computer program product
US8980864B2 (en) 2013-03-15 2015-03-17 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
CA2905410A1 (en) 2013-03-15 2014-09-25 Abbott Molecular Inc. Systems and methods for detection of genomic copy number changes
AU2014237563B2 (en) 2013-03-15 2020-06-11 Becton, Dickinson And Company Detection of neisseria gonorrhoeaes
WO2014144092A1 (en) 2013-03-15 2014-09-18 Nugen Technologies, Inc. Sequential sequencing
EP2971140B1 (en) 2013-03-15 2019-01-16 Ibis Biosciences, Inc. Methods to assess contamination in dna sequencing
CN105392892A (en) 2013-03-27 2016-03-09 六品科技公司 Recombinant phage and bacterial detection methods
EP3457138A3 (en) 2013-04-30 2019-06-19 Université de Montréal Novel biomarkers for acute myeloid leukemia
ES2727898T3 (en) 2013-05-02 2019-10-21 Univ Michigan Regents Deuterated Amlexanox with enhanced metabolic stability
CN104178562B (en) 2013-05-21 2018-11-09 生物梅里埃股份公司 A kind of colorectal cancer prognosis kit
EP3004388B2 (en) 2013-05-29 2023-05-31 Chronix Biomedical Detection and quantification of donor cell-free dna in the circulation of organ transplant recipients
US20160138013A1 (en) 2013-05-30 2016-05-19 The Regents Of The University Of California Substantially unbiased amplification of genomes
CA2898747C (en) 2013-06-13 2021-09-21 Ariosa Diagnostics, Inc. Statistical analysis for non-invasive sex chromosome aneuploidy determination
JP2016521996A (en) 2013-06-19 2016-07-28 サンプルシックス テクノロジーズ,インコーポレイティド Phage-based bacterial detection assay
US9708657B2 (en) 2013-07-01 2017-07-18 Adaptive Biotechnologies Corp. Method for generating clonotype profiles using sequence tags
CN110592244A (en) 2013-07-25 2019-12-20 德诚分子诊断 Methods and compositions for detecting bacterial contamination
US9926597B2 (en) 2013-07-26 2018-03-27 Life Technologies Corporation Control nucleic acid sequences for use in sequencing-by-synthesis and methods for designing the same
EP3033437B1 (en) 2013-08-14 2023-11-08 Gen-Probe Incorporated Compositions and methods for detecting hev nucleic acid
WO2015031691A1 (en) 2013-08-28 2015-03-05 Cellular Research, Inc. Massively parallel single cell analysis
US11545241B1 (en) 2013-09-07 2023-01-03 Labrador Diagnostics Llc Systems and methods for analyte testing and data management
WO2015048744A2 (en) 2013-09-30 2015-04-02 Moderna Therapeutics, Inc. Polynucleotides encoding immune modulating polypeptides
US10323076B2 (en) 2013-10-03 2019-06-18 Modernatx, Inc. Polynucleotides encoding low density lipoprotein receptor
CN105683980B (en) 2013-10-04 2018-08-24 生命科技股份有限公司 The method and system of effect model stage by stage is established in using the sequencing for terminating chemical substance
US11901041B2 (en) 2013-10-04 2024-02-13 Bio-Rad Laboratories, Inc. Digital analysis of nucleic acid modification
JP6525473B2 (en) 2013-11-13 2019-06-05 ニューゲン テクノロジーズ, インコーポレイテッド Compositions and methods for identifying replicate sequencing leads
WO2015083004A1 (en) 2013-12-02 2015-06-11 Population Genetics Technologies Ltd. Method for evaluating minority variants in a sample
US9476853B2 (en) 2013-12-10 2016-10-25 Life Technologies Corporation System and method for forming microwells
US11859246B2 (en) 2013-12-11 2024-01-02 Accuragen Holdings Limited Methods and compositions for enrichment of amplification products
CN104946737B (en) 2013-12-11 2019-02-22 安可济控股有限公司 For detecting the composition and method of rare sequence variants
US11286519B2 (en) 2013-12-11 2022-03-29 Accuragen Holdings Limited Methods and compositions for enrichment of amplification products
US9944977B2 (en) 2013-12-12 2018-04-17 Raindance Technologies, Inc. Distinguishing rare variations in a nucleic acid sequence from a sample
US9909181B2 (en) 2013-12-13 2018-03-06 Northwestern University Biomarkers for post-traumatic stress states
US11193176B2 (en) 2013-12-31 2021-12-07 Bio-Rad Laboratories, Inc. Method for detecting and quantifying latent retroviral RNA species
US20150337364A1 (en) 2014-01-27 2015-11-26 ArcherDX, Inc. Isothermal Methods and Related Compositions for Preparing Nucleic Acids
EP3102691B1 (en) 2014-02-03 2019-09-11 Thermo Fisher Scientific Baltics UAB Method for controlled dna fragmentation
DE102015017080B3 (en) 2014-02-28 2024-01-04 Gen-Probe Incorporated Method for isolating nucleic acid from cytological samples in liquid preservatives containing formaldehyde
WO2015131107A1 (en) 2014-02-28 2015-09-03 Nugen Technologies, Inc. Reduced representation bisulfite sequencing with diversity adaptors
CA2940591C (en) 2014-02-28 2020-11-17 Brett Wolfe KIRKCONNELL Method of isolating nucleic acid from specimens in liquid-based cytology preservatives containing formaldehyde
ES2741740T3 (en) 2014-03-05 2020-02-12 Adaptive Biotechnologies Corp Methods that use synthetic molecules that contain random nucleotide segments
WO2015147370A1 (en) 2014-03-28 2015-10-01 Seegene, Inc. Detection of target nucleic acid sequences using different detection temperatures
EP3125936B1 (en) 2014-03-31 2019-05-08 Debiopharm International SA Fgfr fusions
US10066265B2 (en) 2014-04-01 2018-09-04 Adaptive Biotechnologies Corp. Determining antigen-specific t-cells
US11390921B2 (en) 2014-04-01 2022-07-19 Adaptive Biotechnologies Corporation Determining WT-1 specific T cells and WT-1 specific T cell receptors (TCRs)
WO2015188178A1 (en) 2014-06-06 2015-12-10 The Regents Of The University Of Michigan Compositions and methods for characterizing and diagnosing periodontal disease
US10364458B2 (en) 2014-07-16 2019-07-30 Tangen Biosciences, Inc. Isothermal methods for amplifying nucleic acid samples
ES2880335T3 (en) 2014-09-09 2021-11-24 Igenomx Int Genomics Corporation Methods and compositions for rapid preparation of nucleic acid libraries
US10676787B2 (en) 2014-10-13 2020-06-09 Life Technologies Corporation Methods, systems, and computer-readable media for accelerated base calling
GB2536745B (en) 2014-10-20 2019-06-26 Gen Probe Inc Red Blood cell lysis solution
EP3715455A1 (en) 2014-10-29 2020-09-30 Adaptive Biotechnologies Corp. Highly-multiplexed simultaneous detection of nucleic acids encoding paired adaptive immune receptor heterodimers from many samples
EP3215260B1 (en) 2014-11-03 2020-01-15 Tangen Biosciences Inc. Apparatus and method for cell, spore, or virus capture and disruption
WO2016073768A1 (en) 2014-11-05 2016-05-12 Veracyte, Inc. Systems and methods of diagnosing idiopathic pulmonary fibrosis on transbronchial biopsies using machine learning and high dimensional transcriptional data
US20160168640A1 (en) 2014-11-10 2016-06-16 Genentech, Inc. Therapeutic and diagnostic methods for il-33-mediated disorders
EP3218482A4 (en) 2014-11-11 2018-05-09 Abbott Molecular Inc. Hybridization probes and methods
US10246701B2 (en) 2014-11-14 2019-04-02 Adaptive Biotechnologies Corp. Multiplexed digital quantitation of rearranged lymphoid receptors in a complex mixture
EP3227687A4 (en) 2014-12-05 2018-10-24 Prelude, Inc. Dcis recurrence and invasive breast cancer
AU2016205179B2 (en) 2015-01-09 2021-06-17 Gen-Probe Incorporated Methods and compositions for diagnosing bacterial vaginosis
US10421993B2 (en) 2015-02-11 2019-09-24 Paragon Genomics, Inc. Methods and compositions for reducing non-specific amplification products
US10208339B2 (en) 2015-02-19 2019-02-19 Takara Bio Usa, Inc. Systems and methods for whole genome amplification
JP6620160B2 (en) 2015-02-20 2019-12-11 タカラ バイオ ユーエスエー, インコーポレイテッド Methods for rapid and accurate dispensing, visualization and analysis of single cells
CA2977821A1 (en) 2015-03-16 2016-09-22 Gen-Probe Incorporated Methods and compositions for detecting bacterial nucleic acid and diagnosing bacterial vaginosis
US9708647B2 (en) 2015-03-23 2017-07-18 Insilixa, Inc. Multiplexed analysis of nucleic acid hybridization thermodynamics using integrated arrays
EP3274470B1 (en) 2015-03-25 2023-06-21 Angle Europe Limited Solid phase nucleic acid target capture and replication using strand displacing polymerases
EP3277294A4 (en) 2015-04-01 2018-11-14 Adaptive Biotechnologies Corp. Method of identifying human compatible t cell receptors specific for an antigenic target
EP3283512A4 (en) 2015-04-17 2018-10-03 Distributed Bio Inc Method for mass humanization of non-human antibodies
EP3286338B1 (en) 2015-04-24 2023-11-01 Atila Biosystems Incorporated Amplification with primers of limited nucleotide composition
CA2982467C (en) 2015-04-24 2024-02-13 Becton, Dickinson And Company Multiplex detection of vulvovaginal candidiasis, trichomoniasis and bacterial vaginosis
EP4220645A3 (en) 2015-05-14 2023-11-08 Life Technologies Corporation Barcode sequences, and related systems and methods
EP3303547A4 (en) 2015-06-05 2018-12-19 Miroculus Inc. Air-matrix digital microfluidics apparatuses and methods for limiting evaporation and surface fouling
US10695762B2 (en) 2015-06-05 2020-06-30 Miroculus Inc. Evaporation management in digital microfluidic devices
EP3653728B1 (en) 2015-06-09 2023-02-01 Life Technologies Corporation Methods, systems, compositions, kits, apparatus and computer-readable media for molecular tagging
CN108450003B (en) 2015-06-19 2022-04-01 赛拉预测公司 Biomarker pairs for predicting preterm birth
EP3124619B1 (en) 2015-07-31 2019-03-06 Menicon Co., Ltd Reagents, method and kit for across and within dog breed glaucoma diagnosis
KR102246710B1 (en) 2015-08-17 2021-04-30 쿠라 온콜로지, 인크. Methods of treating cancer patients with farnesyl transferase inhibitors
US10647981B1 (en) 2015-09-08 2020-05-12 Bio-Rad Laboratories, Inc. Nucleic acid library generation methods and compositions
US9499861B1 (en) 2015-09-10 2016-11-22 Insilixa, Inc. Methods and systems for multiplex quantitative nucleic acid amplification
CN108368545B (en) 2015-10-09 2022-05-17 安可济控股有限公司 Methods and compositions for enriching amplification products
ES2899242T3 (en) 2015-11-20 2022-03-10 Seegene Inc Method for calibrating a data set of a target analyte
CN108699505A (en) 2015-12-03 2018-10-23 安可济控股有限公司 It is used to form the method and composition of connection product
WO2017100283A1 (en) 2015-12-09 2017-06-15 Life Technologies Corporation Detection and quantification of nucleic acid molecules associated with a surface
WO2017105104A1 (en) 2015-12-15 2017-06-22 Seegene, Inc. Signal extraction for a target nucleic acid sequence
EP3764079B1 (en) 2015-12-31 2023-08-09 Gen-Probe Incorporated System and method for monitoring the performance of an optical signal detector
AU2017205399C1 (en) 2016-01-04 2023-06-15 Gen-Probe Incorporated Methods and compositions for detecting Candida species
EP3402900A1 (en) 2016-01-12 2018-11-21 Interleukin Genetics, Inc. Methods for predicting response to treatment
US10626383B2 (en) 2016-01-15 2020-04-21 Thermo Fisher Scientific Baltics Uab Thermophilic DNA polymerase mutants
CA3011991A1 (en) 2016-01-21 2017-07-27 T2 Biosystems, Inc. Rapid antimicrobial susceptibility testing using high-sensitivity direct detection methods
WO2017132538A1 (en) 2016-01-29 2017-08-03 The Regents Of The University Of Michigan Amlexanox analogs
KR102300738B1 (en) 2016-02-05 2021-09-10 주식회사 씨젠 How to reduce the noise level of a data set for a target analyte
WO2017155858A1 (en) 2016-03-07 2017-09-14 Insilixa, Inc. Nucleic acid sequence identification using solid-phase cyclic single base extension
US20210002715A1 (en) 2016-04-14 2021-01-07 T2 Biosystems, Inc. Methods and systems for amplification in complex samples
US20190119758A1 (en) 2016-04-22 2019-04-25 Kura Oncology, Inc. Methods of selecting cancer patients for treatment with farnesyltransferase inhibitors
JP7134869B2 (en) 2016-04-27 2022-09-12 ジェン-プローブ・インコーポレーテッド Hemolysis reagent
US10619205B2 (en) 2016-05-06 2020-04-14 Life Technologies Corporation Combinatorial barcode sequences, and related systems and methods
CA3024630A1 (en) 2016-05-16 2017-11-23 Accuragen Holdings Limited Method of improved sequencing by strand identification
US20170351807A1 (en) 2016-06-01 2017-12-07 Life Technologies Corporation Methods and systems for designing gene panels
JP6803930B2 (en) 2016-06-10 2021-01-06 ジェン−プローブ・インコーポレーテッド Compositions and Methods for Detecting ZIKA Viral Nucleic Acids
WO2018013509A1 (en) 2016-07-11 2018-01-18 Arizona Board Of Regents On Behalf Of The University Of Arizona Compositions and methods for diagnosing and treating arrhythmias
CN109070044B (en) 2016-07-21 2021-07-30 宝生物工程(美国)有限公司 Multi-Z-plane imaging and dispensing using multi-aperture device
EP3497245A4 (en) 2016-08-15 2020-06-03 Accuragen Holdings Limited Compositions and methods for detecting rare sequence variants
CA3034064A1 (en) 2016-08-22 2018-03-01 Miroculus Inc. Feedback system for parallel droplet control in a digital microfluidic device
US11414708B2 (en) 2016-08-24 2022-08-16 Decipher Biosciences, Inc. Use of genomic signatures to predict responsiveness of patients with prostate cancer to post-operative radiation therapy
US10704082B2 (en) 2016-09-15 2020-07-07 ArcherDX, Inc. Methods of nucleic acid sample preparation
US10683531B2 (en) 2016-09-15 2020-06-16 ArcherDX, Inc. Methods of nucleic acid sample preparation for analysis of cell-free DNA
WO2018057928A1 (en) 2016-09-23 2018-03-29 Grail, Inc. Methods of preparing and analyzing cell-free nucleic acid sequencing libraries
EP3519554A4 (en) 2016-09-30 2020-05-20 The Governing Council Of The University Of Toronto System for identifying and targeting individual cells within a heterogeneous population for selective extraction of cellular content
WO2018071522A1 (en) 2016-10-11 2018-04-19 Life Technologies Corporation Rapid amplification of nucleic acids
AU2017346854A1 (en) 2016-10-19 2019-05-30 Gen-Probe Incorporated Compositions and methods for detecting or quantifying hepatitis C virus
TW201818965A (en) 2016-11-03 2018-06-01 美商庫拉腫瘤技術股份有限公司 Methods of treating cancer patients with FARNESYLTRANSFERASE inhibitors
WO2018085862A2 (en) 2016-11-07 2018-05-11 Grail, Inc. Methods of identifying somatic mutational signatures for early cancer detection
WO2018094171A1 (en) 2016-11-21 2018-05-24 Gen-Probe Incorporated Compositions and methods for detecting or quantifying hepatitis b virus
US20180163201A1 (en) 2016-12-12 2018-06-14 Grail, Inc. Methods for tagging and amplifying rna template molecules for preparing sequencing libraries
WO2018111835A1 (en) 2016-12-12 2018-06-21 Dana-Farber Cancer Institute, Inc. Compositions and methods for molecular barcoding of dna molecules prior to mutation enrichment and/or mutation detection
US10427162B2 (en) 2016-12-21 2019-10-01 Quandx Inc. Systems and methods for molecular diagnostics
WO2018119399A1 (en) 2016-12-23 2018-06-28 Grail, Inc. Methods for high efficiency library preparation using double-stranded adapters
CN110383061A (en) 2016-12-28 2019-10-25 米罗库鲁斯公司 Digital microcurrent-controlled device and method
IL267836B2 (en) 2017-01-04 2023-09-01 Complete Genomics Inc Stepwise sequencing by non-labeled reversible terminators or natural nucleotides
WO2018127786A1 (en) 2017-01-06 2018-07-12 Oslo Universitetssykehus Hf Compositions and methods for determining a treatment course of action
CN110382716A (en) 2017-01-10 2019-10-25 佰隆基因公司 For reducing the method and composition of the redundancy molecular barcode generated in primer extension reaction
US10329620B2 (en) 2017-01-12 2019-06-25 Cardioforecast Ltd. Methods and kits for treating cardiovascular disease
CA3050984A1 (en) 2017-01-20 2018-07-26 Decipher Biosciences, Inc. Molecular subtyping, prognosis, and treatment of bladder cancer
US9956215B1 (en) 2017-02-21 2018-05-01 Kura Oncology, Inc. Methods of treating cancer with farnesyltransferase inhibitors
KR20200090982A (en) 2017-02-21 2020-07-29 쿠라 온콜로지, 인크. Methods of treating cancer with farnesyltransferase inhibitors
WO2018165600A1 (en) 2017-03-09 2018-09-13 Genomedx Biosciences, Inc. Subtyping prostate cancer to predict response to hormone therapy
CA3055427C (en) 2017-03-24 2022-12-13 Gen-Probe Incorporated Compositions and methods for detection of viral pathogens in samples
CN110573254B (en) 2017-03-24 2022-12-02 简·探针公司 Cap assembly and associated method of use
EP4282985A3 (en) 2017-03-24 2024-02-28 Gen-Probe Incorporated Compositions and methods for detecting or quantifying parainfluenza virus
WO2018183918A1 (en) 2017-03-30 2018-10-04 Grail, Inc. Enhanced ligation in sequencing library preparation
WO2018183897A1 (en) 2017-03-31 2018-10-04 Grail, Inc. Higher target capture efficiency using probe extension
US11584958B2 (en) 2017-03-31 2023-02-21 Grail, Llc Library preparation and use thereof for sequencing based error correction and/or variant identification
US11623219B2 (en) 2017-04-04 2023-04-11 Miroculus Inc. Digital microfluidics apparatuses and methods for manipulating and processing encapsulated droplets
US11708613B2 (en) 2017-05-03 2023-07-25 The United States of America, as Represened by the Secretary, Department of Health and Human Services Rapid detection of Zika virus by reverse transcription loop-mediated isothermal amplification
EP4124862A1 (en) 2017-05-05 2023-02-01 bioMérieux Method for detecting immune cellular response
CA3062716A1 (en) 2017-05-12 2018-11-15 Decipher Biosciences, Inc. Genetic signatures to predict prostate cancer metastasis and identify tumor agressiveness
WO2018213803A1 (en) 2017-05-19 2018-11-22 Neon Therapeutics, Inc. Immunogenic neoantigen identification
US10995104B2 (en) 2017-05-30 2021-05-04 Roche Molecular System, Inc. Catalysts for reversing formaldehyde adducts and crosslinks
US11542540B2 (en) 2017-06-16 2023-01-03 Life Technologies Corporation Control nucleic acids, and compositions, kits, and uses thereof
US11217329B1 (en) 2017-06-23 2022-01-04 Veracyte, Inc. Methods and systems for determining biological sample integrity
WO2019002178A1 (en) 2017-06-26 2019-01-03 Thermo Fisher Scientific Baltics Uab Thermophilic dna polymerase mutants
CA3068994C (en) 2017-07-10 2023-06-27 Gen-Probe Incorporated Analytical systems and methods for nucleic acid amplification using sample assigning parameters
WO2019023133A1 (en) 2017-07-24 2019-01-31 Miroculus Inc. Digital microfluidics systems and methods with integrated plasma collection device
US11174511B2 (en) 2017-07-24 2021-11-16 Dana-Farber Cancer Institute, Inc. Methods and compositions for selecting and amplifying DNA targets in a single reaction mixture
EP3664804A4 (en) 2017-08-07 2021-04-14 Kura Oncology, Inc. Methods of treating cancer with farnesyltransferase inhibitors
US10806730B2 (en) 2017-08-07 2020-10-20 Kura Oncology, Inc. Methods of treating cancer with farnesyltransferase inhibitors
US11859257B2 (en) 2017-08-11 2024-01-02 Gen-Probe Incorporated Compositions and methods for detecting Staphylococcus aureus
EP3668996A4 (en) 2017-08-18 2021-04-21 Sera Prognostics, Inc. Pregnancy clock proteins for predicting due date and time to birth
JP7341124B2 (en) 2017-09-01 2023-09-08 ミロキュラス インコーポレイテッド Digital microfluidic device and its usage
US20200263170A1 (en) 2017-09-14 2020-08-20 Grail, Inc. Methods for preparing a sequencing library from single-stranded dna
EP4269583A3 (en) 2017-09-28 2024-01-17 Grail, LLC Enrichment of short nucleic acid fragments in sequencing library preparation
US11099202B2 (en) 2017-10-20 2021-08-24 Tecan Genomics, Inc. Reagent delivery system
CN111465707A (en) 2017-11-17 2020-07-28 简·探针公司 Compositions and methods for detecting C1orf43 nucleic acids
US20200318171A1 (en) 2017-12-15 2020-10-08 Gen-Probe Incorporated Compositions and Methods for Detecting Toxigenic Clostridium Difficile
US11414656B2 (en) 2017-12-15 2022-08-16 Grail, Inc. Methods for enriching for duplex reads in sequencing and error correction
US20190237161A1 (en) 2017-12-22 2019-08-01 Grail, Inc. Error removal using improved library preparation methods
WO2019133727A1 (en) 2017-12-29 2019-07-04 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Universal influenza virus probe set for enrichment of any influenza virus nucleic acid
JP2021512303A (en) 2018-01-29 2021-05-13 ジェン−プローブ・インコーポレーテッド Analytical systems and methods
US11814674B2 (en) 2018-02-02 2023-11-14 The United States Of America, As Represented By The Secretary Department Of Health And Human Services Random amplification methods for extremely low input nucleic acids
KR20230142806A (en) 2018-02-09 2023-10-11 제넨테크, 인크. Therapeutic and diagnostic methods for mast cell-mediated inflammatory diseases
US11203782B2 (en) 2018-03-29 2021-12-21 Accuragen Holdings Limited Compositions and methods comprising asymmetric barcoding
FR3080185A1 (en) 2018-04-16 2019-10-18 Biomerieux EVALUATION OF THE RISK OF COMPLICATION IN A PATIENT SUSPECTED TO HAVE AN INFECTION HAVING A SOFA SCORE LESS THAN TWO
CN112654721A (en) 2018-06-13 2021-04-13 简·探针公司 Compositions and methods for detecting group B streptococcal nucleic acids
EP3814496B1 (en) 2018-06-28 2023-11-22 Gen-Probe Incorporated Sample preparation method and system
EP3814532A1 (en) 2018-06-29 2021-05-05 bioMérieux Method for determining in vitro or ex vivo the immune status of an individual
FR3085689A1 (en) 2018-09-12 2020-03-13 Biomerieux PROCESS FOR DETERMINING IN VITRO OR EX VIVO THE IMMUNE STATUS OF AN INDIVIDUAL
US20210317515A1 (en) 2018-07-10 2021-10-14 Gen-Probe Incorporated Methods and systems for detecting and quantifying nucleic acids
CN112714796A (en) 2018-07-27 2021-04-27 芝加哥大学 Method for amplifying bisulfite-treated DNA
SG11202101934SA (en) 2018-07-30 2021-03-30 Readcoor Llc Methods and systems for sample processing or analysis
WO2020028631A1 (en) 2018-08-01 2020-02-06 Gen-Probe Incorporated Compositions and methods for detecting nucleic acids of epstein-barr virus
US20210310059A1 (en) 2018-08-08 2021-10-07 Gen-Probe Incorporated Compositions, methods and kits for detecting mycoplasma genitalium
AU2019326462A1 (en) 2018-08-21 2021-03-25 Gen-Probe Incorporated Compositions and methods for amplifying, detecting or quantifying human cytomegalovirus
EP3841222A1 (en) 2018-08-24 2021-06-30 Gen-Probe Incorporated Compositions and methods for detecting bacterial nucleic acid and diagnosing bacterial vaginosis
AU2019339508A1 (en) 2018-09-14 2021-04-15 Prelude Corporation Method of selection for treatment of subjects at risk of invasive breast cancer
AU2019350777A1 (en) 2018-09-27 2021-05-20 Gen-Probe Incorporated Compositions and methods for detecting Bordetella pertussis and Bordetellaparapertussis nucleic acid
US20220017980A1 (en) 2018-10-01 2022-01-20 Gen-Probe Incorporated Compositions and methods for amplifying or detecting varicella-zoster virus
CN113195743A (en) 2018-10-22 2021-07-30 简·探针公司 Compositions and methods for amplifying, detecting or quantifying human polyoma virus BK virus
TW202031259A (en) 2018-11-01 2020-09-01 美商庫拉腫瘤技術股份有限公司 Methods of treating cancer with farnesyltransferase inhibitors
CA3122494A1 (en) 2018-12-13 2020-06-18 Dna Script Direct oligonucleotide synthesis on cells and biomolecules
US11512349B2 (en) 2018-12-18 2022-11-29 Grail, Llc Methods for detecting disease using analysis of RNA
WO2020142347A2 (en) 2018-12-31 2020-07-09 Gen-Probe Incorporated Systems and methods for filling multi-well cartridges with solid reagents
JP2022515912A (en) 2019-01-03 2022-02-22 ディーエヌエー スクリプト One-pot synthesis of oligonucleotide sets
WO2020180663A1 (en) 2019-03-01 2020-09-10 Kura Oncology, Inc. Methods of treating cancer with farnesyltransferase inhibitors
EP3937780A4 (en) 2019-03-14 2022-12-07 InSilixa, Inc. Methods and systems for time-gated fluorescent-based detection
EP3708678A1 (en) 2019-03-15 2020-09-16 Adisseo France S.A.S. Process for identifying a stress state in a subject
US20220098647A1 (en) 2019-03-22 2022-03-31 Gen-Probe Incorporated Compositions and Methods for Detecting Group A Streptococcus
CN113646007A (en) 2019-03-29 2021-11-12 希森美康株式会社 Novel artificial nucleic acid, method for producing same, and use thereof
WO2020205486A1 (en) 2019-03-29 2020-10-08 Kura Oncology, Inc. Methods of treating squamous cell carcinomas with farnesyltransferase inhibitors
WO2020205387A1 (en) 2019-04-01 2020-10-08 Kura Oncology, Inc. Methods of treating cancer with farnesyltransferase inhibitors
CN114206499A (en) 2019-04-08 2022-03-18 米罗库鲁斯公司 Multi-cartridge digital microfluidic devices and methods of use
US20220305001A1 (en) 2019-05-02 2022-09-29 Kura Oncology, Inc. Methods of treating acute myeloid leukemia with farnesyltransferase inhibitors
CA3176696A1 (en) 2019-05-03 2020-11-12 Gen-Probe Incorporated Receptacle transport system for an analytical system
CA3142662A1 (en) 2019-06-06 2020-12-10 Sitokine Limited Compositions and methods for treating lung, colorectal and breast cancer
US20220307093A1 (en) 2019-07-03 2022-09-29 Gen-Probe Incorporated Oligonucleotides for use in determining the presence of trichomonas vaginalis in a sample
WO2021016614A1 (en) 2019-07-25 2021-01-28 Miroculus Inc. Digital microfluidics devices and methods of use thereof
WO2021028469A1 (en) 2019-08-12 2021-02-18 Sitokine Limited Compositions and methods for treating cytokine release syndrome and neurotoxicity
EP4018004A1 (en) 2019-08-23 2022-06-29 Gen-Probe Incorporated Compositions, methods and kits for detecting treponema pallidum
CA3153514A1 (en) 2019-09-05 2021-03-11 Gen-Probe Incorporated Detection of chlamydia trachomatis nucleic acid variants
FR3101358A1 (en) 2019-09-27 2021-04-02 Bioaster Method for determining the risk of occurrence of an infection associated with care in a patient
FR3101423A1 (en) 2019-10-01 2021-04-02 bioMérieux Method for determining the ability of an individual to respond to a stimulus
CN114729402A (en) 2019-10-01 2022-07-08 生物梅里埃公司 Method for determining the ability of an individual to respond to a stimulus
JP2023503946A (en) 2019-12-09 2023-02-01 ジェン-プローブ・インコーポレーテッド Quantification of polynucleotide analytes from dried samples
US20230227924A1 (en) 2019-12-23 2023-07-20 Abbott Laboratories Compositions and methods for detecting picobirnavirus
WO2021138325A1 (en) 2019-12-30 2021-07-08 Abbott Laboratories Compositions and methods for detecting bunyavirus
CA3167112A1 (en) 2020-01-16 2021-07-22 Dnae Diagnostics Limited Compositions, kits and methods for isolating target polynucleotides
CA3174532A1 (en) 2020-03-04 2021-09-10 Gen-Probe Incorporated Compositions and methods for detecting sars-cov-2 nucleic acid
IL296319A (en) 2020-03-09 2022-11-01 Janssen Biotech Inc Compositions and methods for quantifying integration of recombinant vector nucleic acid
WO2021180858A1 (en) 2020-03-13 2021-09-16 Medimmune Limited Therapeutic methods for the treatment of subjects with risk alelles in il33
WO2021205013A1 (en) 2020-04-09 2021-10-14 Sitokine Limited Compositions and methods for treating covid-19
US10941453B1 (en) 2020-05-20 2021-03-09 Paragon Genomics, Inc. High throughput detection of pathogen RNA in clinical specimens
WO2021252574A1 (en) 2020-06-10 2021-12-16 Sera Prognostics, Inc. Nucleic acid biomarkers for placental dysfunction
FR3112210A1 (en) 2020-07-06 2022-01-07 bioMérieux Method for determining the risk of occurrence of an infection associated with care in a patient
FR3112207A1 (en) 2020-07-06 2022-01-07 bioMérieux Method for determining the risk of occurrence of an infection associated with care in a patient
FR3112211A1 (en) 2020-07-06 2022-01-07 bioMérieux Method for determining the risk of occurrence of an infection associated with care in a patient
FR3112208A1 (en) 2020-07-06 2022-01-07 bioMérieux Method for determining the risk of occurrence of an infection associated with care in a patient
FR3112209A1 (en) 2020-07-06 2022-01-07 bioMérieux Method for determining the risk of complications in a patient
US20230243001A1 (en) 2020-07-17 2023-08-03 Gen-Probe Incorporated Detection of Macrolide-Resistant Mycoplasma Genitalium
WO2022056078A1 (en) 2020-09-11 2022-03-17 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Rnase h-assisted detection assay for rna (radar)
JPWO2022065413A1 (en) 2020-09-25 2022-03-31
WO2022087257A2 (en) 2020-10-21 2022-04-28 Gen-Probe Incorporated Fluid container management system
AU2021376490A1 (en) 2020-11-05 2023-05-11 Becton, Dickinson And Company Rapid identification and typing of vibrio parahaemolyticus
AU2021374695A1 (en) 2020-11-05 2023-07-13 Becton, Dickinson And Company Multiplex detection and typing of vibrio cholerae
JP2023547536A (en) 2020-11-05 2023-11-10 ベクトン・ディキンソン・アンド・カンパニー Multiplex detection of bacterial respiratory pathogens
WO2022106795A1 (en) 2020-11-20 2022-05-27 bioMérieux Method for classifying an individual
WO2022241291A1 (en) 2021-05-14 2022-11-17 Gen-Probe Incorporated Compositions and methods for detecting human adenovirus nucleic acid
AU2022297174A1 (en) 2021-06-24 2024-02-08 Seegene, Inc. Automated analysis system using individually operated biological devices, analysis method and storage medium
AU2022304654A1 (en) 2021-07-01 2023-12-14 Gen-Probe Incorporated Enzyme formulations and reaction mixtures for nucleic acid amplification
FR3125065A1 (en) 2021-07-08 2023-01-13 bioMérieux Method and kit for detecting a replicative respiratory virus
AU2022307677A1 (en) 2021-07-09 2024-01-25 Cepheid High-level multiplexing reaction vessel, reagent spotting device and associated methods
GB202110479D0 (en) 2021-07-21 2021-09-01 Dnae Diagnostics Ltd Compositions, kits and methods for sequencing target polynucleotides
AU2022315102A1 (en) 2021-07-21 2024-02-22 Seegene, Inc. Assembled analysis system, method, and computer readable recording medium
WO2023002203A1 (en) 2021-07-21 2023-01-26 Dnae Diagnostics Limited Method and system comprising a cartridge for sequencing target polynucleotides
GB202110485D0 (en) 2021-07-21 2021-09-01 Dnae Diagnostics Ltd Compositions, kits and methods for sequencing target polynucleotides
WO2023010008A1 (en) 2021-07-27 2023-02-02 Gen-Probe Incorporated Compositions and methods for detecting gastrointestinal pathogens
WO2023021330A1 (en) 2021-08-16 2023-02-23 University Of Oslo Compositions and methods for determining a treatment course of action
US20230121442A1 (en) 2021-10-06 2023-04-20 Johnson & Johnson Consumer Inc. Method of Quantifying Product Impact on Human Microbiome
US11857961B2 (en) 2022-01-12 2024-01-02 Miroculus Inc. Sequencing by synthesis using mechanical compression
WO2023152568A2 (en) 2022-02-10 2023-08-17 Oslo Universitetssykehus Hf Compositions and methods for characterizing lung cancer
WO2023175434A1 (en) 2022-03-15 2023-09-21 Diagenode S.A. Detection of methylation status of a dna sample
US11680293B1 (en) 2022-04-21 2023-06-20 Paragon Genomics, Inc. Methods and compositions for amplifying DNA and generating DNA sequencing results from target-enriched DNA molecules
EP4282980A1 (en) 2022-05-23 2023-11-29 Mobidiag Oy Methods for amplifying a nucleic acid
WO2024003332A1 (en) 2022-06-30 2024-01-04 F. Hoffmann-La Roche Ag Controlling for tagmentation sequencing library insert size using archaeal histone-like proteins
WO2024015331A1 (en) 2022-07-12 2024-01-18 Genentech, Inc. Therapeutic and diagnostic methods for multiple sclerosis

Family Cites Families (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA559709A (en) * 1958-07-01 International Standard Electric Corporation Travelling wave tubes
US4957858A (en) * 1986-04-16 1990-09-18 The Salk Instute For Biological Studies Replicative RNA reporter systems
US4786600A (en) * 1984-05-25 1988-11-22 The Trustees Of Columbia University In The City Of New York Autocatalytic replication of recombinant RNA
US4683195A (en) * 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683202A (en) * 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
AU606043B2 (en) * 1985-03-28 1991-01-31 F. Hoffmann-La Roche Ag Detection of viruses by amplification and hybridization
US4800159A (en) * 1986-02-07 1989-01-24 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences
EP0276302B1 (en) * 1986-08-11 1993-04-28 Siska Diagnostics,Inc. Nucleic acid probe assay methods and compositions
DE3628485A1 (en) * 1986-08-22 1988-02-25 Iro Ab DEVICE FOR STORING YARN
IL86724A (en) * 1987-06-19 1995-01-24 Siska Diagnostics Inc Method and kits for the amplification and detection of nucleic acid sequences
US4994368A (en) * 1987-07-23 1991-02-19 Syntex (U.S.A.) Inc. Amplification method for polynucleotide assays
JP2774121B2 (en) * 1987-07-31 1998-07-09 ザ ボード オブ トラスティーズ オブ ザ リーランド スタンフォード ジュニア ユニバーシティ Selective amplification of target polynucleotide sequence
EP0638807B1 (en) * 1987-09-21 2002-07-17 Gen-Probe Incorporated Protection assay
CA1340807C (en) * 1988-02-24 1999-11-02 Lawrence T. Malek Nucleic acid amplification process
US5130238A (en) * 1988-06-24 1992-07-14 Cangene Corporation Enhanced nucleic acid amplification process
KR0148265B1 (en) * 1988-12-16 1998-10-15 에프.지이.엠 헤르만스 Self-sustained sequence replication system
CA2016553A1 (en) * 1989-05-16 1990-11-16 Dyann F. Wirth Dna hybridization probes for identification of mycobacteria
US5112734A (en) * 1989-05-26 1992-05-12 Gene-Trak Systems Target-dependent synthesis of an artificial gene for the synthesis of a replicatable rna
CA2020958C (en) * 1989-07-11 2005-01-11 Daniel L. Kacian Nucleic acid sequence amplification methods
AU650622B2 (en) * 1989-07-11 1994-06-30 Gen-Probe Incorporated Nucleic acid sequence amplification methods utilizing a transcription complex
FR2659086B1 (en) * 1990-03-02 1992-06-12 Pasteur Institut NUCLEOTIDE FRAGMENTS CHARACTERISTICS OF DNA FRAGMENTS OF MYCOBACTERIA. THEIR USE AS A PRIMER FOR THE DETECTION OF AN INFECTION DUE TO MYCOBACTERIA.
FR2651505B1 (en) * 1989-09-06 1994-07-22 Pasteur Institut FRAGMENTS OF NUCLEIC ACIDS DERIVED FROM AN APPROPRIATE MYCOBACTERIA GENOME, THEIR APPLICATIONS IN THE DIAGNOSIS OF MYCOBACTERIA AND PLASMID INFECTIONS CONTAINING SAID FRAGMENTS.
AU7653691A (en) * 1990-04-05 1991-10-30 United States of America, as represented by the Secretary, U.S. Department of Commerce, The Modified rna template-specific polymerase chain reaction
FR2663033B1 (en) * 1990-06-08 1992-09-04 Pasteur Institut SPECIFIC DETECTION OF MYCOBACTERIUM TUBERCULOSIS.

Also Published As

Publication number Publication date
US5399491A (en) 1995-03-21
US5824518A (en) 1998-10-20
CA2020958A1 (en) 1991-01-12
US5888779A (en) 1999-03-30

Similar Documents

Publication Publication Date Title
CA2020958C (en) Nucleic acid sequence amplification methods
EP0731174B1 (en) Nucleic acid sequence amplification methods
EP0587298B1 (en) Nucleic acid sequence amplification method
US5766849A (en) Methods of amplifying nucleic acids using promoter-containing primer sequence
US6063604A (en) Target nucleic acid sequence amplification
US5888729A (en) Oligonucleotide probes and methods for detecting Streptococcus pneumoniae
US7083922B2 (en) Detection of HIV
US7009041B1 (en) Oligonucleotides for nucleic acid amplification and for the detection of Mycobacterium tuberculosis
AU711448B2 (en) Oligonucleotide probes and methods for detecting streptococcus pneumoniae

Legal Events

Date Code Title Description
EEER Examination request
MKEX Expiry