CA2221467A1 - Nucleic acid detection methods - Google Patents
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- CA2221467A1 CA2221467A1 CA002221467A CA2221467A CA2221467A1 CA 2221467 A1 CA2221467 A1 CA 2221467A1 CA 002221467 A CA002221467 A CA 002221467A CA 2221467 A CA2221467 A CA 2221467A CA 2221467 A1 CA2221467 A1 CA 2221467A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6823—Release of bound markers
Abstract
The invention relates to methods for rapidly determining the sequence and/or length of a target sequence. The target sequence may be a series of known or unknown repeat sequences which are hybridized to an array of probes. The hybridized array is digested with a single-strand nuclease and free 3'-hydroxyl groups extended with a nucleic acid polymerase. Nuclease cleaved heteroduplexes can be easily distinguished from nuclease uncleaved heteroduplexes by differential labeling. Probes and target can be differentially labeled with detectable labels. Matched target can be detected by cleaving resulting loops from the hybridized target and creating free 3-hydroxyl groups. These groups are recognized and extended by polymerases added into the reaction system which also adds or releases one label into solution.
Analysis of the resulting products using either solid phase or solution. These methods can be used to detect characteristic nucleic acid sequences, to determine target sequence and to screen for genetic defects and disorders.
Assays can be conducted on solid surfaces allowing for multiple reactions to be conducted in parallel and, if desired, automated.
Analysis of the resulting products using either solid phase or solution. These methods can be used to detect characteristic nucleic acid sequences, to determine target sequence and to screen for genetic defects and disorders.
Assays can be conducted on solid surfaces allowing for multiple reactions to be conducted in parallel and, if desired, automated.
Description
CA 02221467 1997-ll-18 W O96/36731 PCTrUS96/06527 NUCLEIC ACID DETECTION METHODS
Ri~hts In The Invention This invention was made with United States Gov~ support 5 under grant llulllbel DE-FG02-93ER61609, awarded by the United States Department of Energy, and grant number AIBS2154, awarded by the United States Depallmenl of the Army, and the United States Go-vellllllen~ has certain rights in the invention.
BackFround of the Invention 10 1. Field of the Invention This invention relates to methods for the detection of repeated and other j~lentifi~hle nucleic acid sequences. The invention also relates to methods for identifying and mapping specific nucleic acid sequences in complex backgrounds.
15 2. Descli~ion of the Background Historically, the ~ gn~ si~ of genetic disease has depended on the irl.s~.lir~lion of abnormal gene products or their clinical effects such as ~n-omi~, mental retardation and certain sch-~hrenia. Through direct analysis of the genome, it is possible to identify genetic mllt~ti~ns and offer tre~tm~nt before20 the ~ ir~ lion of ~ylll~OlllS. Genetic analyses ~elÇoll,led today range from gross analysis such as karyotyping to the analysis of individual base pairs by sequencing. Although a great deal of progress has been made, nucleic acid sequencing is still too labor hllel~ive and e~-~ensive for everyday diagnosis beyond the experimental m~ 1 research laboratory.
Many genetic defects such as Burkett's Iymphoma and some sickle cell anemia and th~ semi~ mutations are ~letect~hle without the use of sequencing. Such techniques include restriction fragment length polymorphism Q (RFLP) and chromosome karyotyping. However, general applicability of these methods is limited as most genetic defects are more modest and do not alter 30 restriction sites or cause chromosome rearrangements. Polymerase chain WO96/36731 PCTrUS96/06S27 reaction and ligase chain reaction can increase the sensitivity of many detection methods and detect single base pair changes in nucleic acid. However, if the mnt~tion involves repeated sequences, the degeneracy of the repeated sequence makes even PCR and LCR detections unreliable.
S Dinucleotide and trinucleotide repeat sequences are increasingly beco~ lg important in genetic analysis. These repeats are both polymorphic and widespread in the human genome and offer a convenient means for locating genes associated with particular phenotypes (M.S. Wehnert et al. Nuc. Acids Res. 22:1701-4, 1994; G. Benson et al., Nuc. Acids Res. 22:4828-36, 1994).
Trinucleotide repeat expansion mutations have been ic1entifie~1 in at least four human genetic ~ e~e,s (C.T. Caskey et al., Sci. 256:784-89, 1992). Each are caused by mutational m.-ch~ni.~m~ whereby normally polymorphic exonic trinucleotide repeats expand beyond the normal size range and alter gene eA~l~,s~ion, mRNA stability or gain certain functions. In FragileX ~yll~ e (FraX; D.L. Nelson et al., Nature Genetics 4:107-108, 1993), the second most col...l~ll genetic form of mental retardation, and also in myotonic dy~ ophy (MD; D.J. Brook et al., Cell 68:799-808, 1992), the repeat e~p~n~ion can be quite large resl-lting n thousands of triplets. In spinal and bulbar mllecul~r atrophy ~SBMA or K~nnPAy disease) and ~llntington's Disease (HD), 20 the exp~n~ion may only consist of twice the normal compliment of repeats.
The genetic element expanded in Fragile X is a triplet called FMR-l. This sequence, CGG, is highly polymorphic in the general population ranging from between about 6 to about 42 triplets per person. Unaffected family members can contain up to 50 repeats. Between 50 and 200, individuals are 25 considered to be pre-mutation. Expansions of several thousand are known to occur in affected patients.
W O96/36731 PCTrUS96/06527 Myotonic dystrophy is an autosomal dominant disorder characteri_ed by muscle we~kness and is the single-most common form of adult ~ onset. The gene responsible, DM-1 has been i~entified There are many methods for ~letecting differences in repeat 5 num'oer. Conventional analyses involve electrophoretic fracti~nation steps. Such steps are seriously limiting in terrns of time and expense and lack the se~ iviLy for detecting short deletions in long sequences (M.B. White et al., Genomics 5:301-6, 1992). Ch~-mic~l detection and cleavage of mi~m~tches, though effective, generally relies on the use of dangerous compounds (P.M. Smooker 10 et al., Mutant. Res. 288:65-77, 1993). The advent of efficient coupling of DNA
to solid surfaces as well as progress in effective flc lcscell~ labeling and ~lett-~tion have paved the way for the development of assays able to detel~ le the length of these dinucleotide and trinucleotide repeats quickly and accurately.
Slllnl l l~ of the Invention The invention overcomes the problems and disadvantages associated with current strategies and designs and provides novel methods for the detection and id~ i ri~ ion of nucleic acid sequences and novel arrays whichcan be ~tili7PA with these methods.
One emb~lim~ont of the invention is directed to methods for 20 ~eL~ g a target seqllen~e within a nucleic acid. The nucleic acid is hybridi_ed to an array of probes wherein each probe com~lises a 5'-region complemlont~ry to the nucleic acid, a 3'-region complem~nt~ry to the nucleic acid, and an internal variable region. The hybridi_ed array is digested with a single-strand specific nuclease and treated with a nucleic acid polymerase. The target 25 sequence may vary in length or sequence, for exarnple, comprising a pluralityof short repeat sequences or a homologous sequence of bases of variable lengths.The sequence and length of the target can be identified by hybridization to a specific probe and resistance to the single-strand specif1c nuclease.
W O96/36731 PCTrUS96/06527 Another embodiment of the invention is directed to -nethods for d~ ing the length of a target sequence within a nucleic acid. A nucleic acid is hybridized to an array of probes wherein each probe comprises a 5'-region complementary to the nucleic acid, a 3'-region complementary to the nucleic S acid, and an internal variable region. The hybridized array is digested with asingle-strand specific nuclease and treated with a nucleic acid polymerase. The nucleic acid may be a PCR product, such as an amplified nucleic acid sequenre, or a DNA or RNA macromolecule purified, if n~-cess~ry, directly from a biological sample. The internal variable region may comprises a homologous 10 sequence of bases such as a sequence inosine residues which non-specifically hybridize to nucleic acids. Hybridized probes resistant to nllrl~e digestion will be the same length as the target sequence.
Another embc~lim~ont of the invention is directed to methods for drL~ .1l;. .;. ~ the number of repeat seq~onres within a nucleic acid. The nucleic 15 acid is hybridized to an array of probes wherein each probe comprises a 5'-region complem~o-nf~ry to the nucleic acid, a 3'-region complem~ont~ry to the nucleic acid, and an internal region which contains one or more repeat sequences. The hybridized array is digested with a single-strand specific mlrlto~e and treated with a nucleic acid polymerase. Hybridized probes rt;:j~L~lL
20 to the nuclease digestion contain the same number of repeats as the target sequence.
Another embodiment of the invention is directed to methods for screening a patient suspected of having a genetic disorder. A tissue sample is obtained from the patient and a nucleic acid sequence obtained by, for example, 25 PCR amplification or direct purification of a target sequence. The nucleic acid is hybridized to an array of probes wherein each probe comprises a 5'-region and a 3'-region, each complementary to the nucleic acid and a variable internal region. The hybridized array is digested with a single-strand specific nuclease and treated with a nucleic acid polymerase. Hybridized ~robes resistant to nuclease digestion will contain a specific number of repeat sequences. The plesellce or absence of the genetic disorder can be deterrnined from the ~ be of repeat sequences which are present.
Another embodiment of the invention is directed to arrays of probes wherein each probe comprises a con~t~nt 5'-region, a constant 3'-region and a variable internal region wherein the variable region comprises one or morerepeat sequences. The repeat sequence comprises heterologous or homologous sequences which are variable in length or base sequence. Sequences contain purine or pyrimidine bases or neutral bases such as inosine. Either the nucleic acids or the probes of the array may be labeled with a d~tect~ble label or fixedto a solid support.
Other emb~lim~onf~ and advantages of the invention are set forth, in part, in the description which follows and, in part, will be obvious from this description and may be lc~nltoA from the practice of the invention.
Descri~tion of th~ Drawin*.~
Figure 1 Sch~m~ti~ of the reaction strategy.
Figure 2 Results of micm~tl~h cleavage with S1 nuclease.
Figure 3 Labeling of S1 cleavage products with radio-labeled nucleotides.
Figure 4 DNA polymerase radiolabeling of Sl cleaved m~tch~l and mi~m~trh~cl substrates.
Figure S .Sch.om~ for detection of mi~m~tch~s using anchored single-stranded oligonucleotide probes.
Figure 6 Two dimensional array for the detection of between 10 to 109 repeats.
Pescription of the Invention As embodied and broadly described herein, the present invention is directed to methods for the detection and i-lPntifi~tion of target sequences by size or base sequence and to arrays of nucleic acid probes which can be utilizedwith these methods.
Nucleic acid screening is widely utilized to detect and identify nucleic acids. The presence or absence of these specific nucleic acids, as S identified by their sequences, can often be considered as evidence of disorders such as infections, neoplasms and genetic diseases. Although there are a wide variety of methods ~;~nlenlly available, sequence detection is generally a slow and expensive proposition requiring costly supplies and the skills of highly trained individuals.
It has been discovered that by combining certain microchemical tools such as nucleic acid probes, nucleic acid hybridization and enzymatic cleavage of heteroduplexed hybrids, procedures can be designed to detect specific target seqllenrPc. Char~cte-ri~tir sequences such as occurs in variations between strains of microorg~ni~m~ and between numbers of repeat sequences can 15 be rapidly and accurately AetectrA and iArntifird Nucleic acids co..l;.i.-i..g these target se~ ellces can be hybridized to oligonucleotide probes that contain seclllenre variations such as a differentrepeat lengths. Loop structures formed by ...ic..~ r,r~ repeats can be cleaved by inrllb~tion with a mlrle~e to geu~,ldle nicked double strands. These nicks 20 are recognized by a nucleic acid polymerase which breaks down or displace one of the strands. Analysis of the products using, for example, dirr~ ial labeling, reveals the nature of the ...i';...~lch as well as the length of the perfectly m~tc'nt-A repeats. As reactions can be co. AIlcte~ in situ and all under the same conditions, process steps can be easily ~uL~ll~d. Many assays could be run in 25 parallel allowing for rapid analysis of target sequence from a variety of sources.
One embodiment of the invention is directed to a method for A~tecting a target sequence within a nucleic acid. Nucleic acids cont~ining target sequences to be ~Tetect~A can be obtained directly or indirectly from natural or W O96/36731 PCTrUS96/06527 ~yllLll~Lic sources. Synthetic sources include sequences chemically synthesized such as oligonucleotides or sequences of PNA. Natural sources of nucleic acid sequences include samples of bodily tissues or fluids obtained from a patient, samples from the environment such as a biomass, soil or body of water. Nucleic acids directly obtained from such sources can be purified, if npcess~ry~ by techniques such as centrifugation, chromatography, chemical extraction, i~lion or other techniques or combinations of techniques known to those of ordinary skill in the art. As sequence information is easily transcribed or replicated, the nucleic acid may be either RNA or DNA and may exist in either the sense or anti-sense orientation.
Nucleic acids are preferably single-stranded, but may be partially single-stranded and partially double-str~n-l~ Single-stranded regions hybridize to probe sequences and double-strand regions can contain recognitions sites for restriction enzymes or other nucleic acid modifying enzymes sites, or used to ch~ lly couple ~let~t~hle labels. If n~cecc~ry, single-stranded nucleic acids can easily be ~r~aLed from target sequences by a number of methods. The strands of most double helixes, once denatured by ~lc~ with 8M urea, low or high pH or 95~C heat, can be s~alal~d by, for example, del~Luli lg electrophoresis. ~lt~rn~tively, polymerase chain reaction using one or an excessof one primer may be performed using the target seq~enre as a template causing the product to consist mainly of one strand. Elongation products formed, for example, using a biotinylated primer can be isolated with a streptavidin column.mRNA, or single stranded cDNA may also be isolated and used as a single stranded target.
The nucleic acid cont~ining the target sequence is preferably generated as a polymerase chain reaction (PCR) product. The basic PCR
process is described in U.S. Patent No. 4,683,195. Variations of the PCR
process are described in U.S. Patent Nos. 5,043,272, 5,057,410 and 5,106,727.
CA 02221467 1997-ll-18 W 096/36731 PCTtUS96tO6527 As a PCR product, the nucleic acid will possess both 5' and 3' terminal sequences which are i(lentical to the sequences of the primers used in the PCR
reaction. These primers flank the seq~ e to be ~mplifi~i which conl~lises the target sequence. Primers are typically less than about 35 nucleotides in length,5 but may be smaller or larger as n~s.~ry to generate the nucleic acid. Althoughnot required, the sequences of the primers are generally known for the primers to specifically hybridize to a relatively unique portion of nucleic acid and generate an j(l~ntifi~hle nucleic acids on PCR amplification. PCR products can be of most any length and can be distinguished from non-specific and undesired 10 amplification products by size.
In PCR and any polymerase ~mplifi(~tion procedure, extensions may be added to the S'-termini of a primer to permit post-amplification manipulations of the product without si~nific~ntly effecting the amplification reaction. These S' extensions may be restriction enzyme recognition-sites, 15 structural sequences or other seqllen~ec desirable for the process. Briefly, template DNA is first denatured by heating in the presence of a large molar excess of each of the two oligonucleotides and the four dNTPs. The reaction mixture is cooled to a temperature that allows the oligonucleotide primer to anneal to target sequences, after which the ~nn~lP~l primers are extended with 20 DNA polymerase. The cycle of dellaluldLion, ~nnt-~ling, and DNA synthesis, the principal of PCR amplification, is repeated many times to generate large qu~nfiti~s of product which can be easily identified. This tt~ e~ture cycling is made possible by the use of a DNA polymerase that does is not destroyed at the higher temperatures required for dellaLul~lion. Nucleic acid polymerases 25 which can be use~ for amplification include both DNA and RNA polymerases Many useful thermostable polymerases for PCR amplification are commercially available such as Taq DNA polymerase (Stratagene; La Jolla, CA) and AmpliTaq DNA polymerase (Perkin-Elmer Cetus; Norwalk, CT).
W O96/36731 PCT~US96/06527 The major product of this exponential reaction is a segment of double stranded nucleic acid, easily converted to single strands by, for example, rhPmir~l, pH or heat del~Lu~ilLion, whose termini are defined by the 5' termini of the oligonucleotide primers and whose length is defined by the fli~t~nre 5 between the primers. Under normal reaction conditions, the amount of polymerase becomes limiting after 25 to 30 cycles or about one million fold amplification. Further amplifir?tion is achieved by diluting the sample 1000 fold and using it as the template for further rounds of amplification in anotherPCR. By this method, amplification levels of 109 to 101~ can be achieved during 10 the course of 60 sequential cycles. This allows the detection, by hybridization with r~-iio~r~ive probes, of a single copy of the target sequence in the presence co..~;....i.-~l;..g DNA. Without the use of seq~lPnti~l PCR, the practical ~letPcti- n limit of PCR can be as low as 10 copies of DNA per sample.
Although PCR is a reliable method for amplifir~tion of target 15 sequences, a number of other techniques can be used such as isothermic amplification, ligase chain reaction (LCR), self s--ct~inPA seq~nre replication (3SR), polymerase chain reaction linked ligase chain reaction (pLCR), gaped ligase chain reaction (gLCR), ligase chain detection (LCD). The principle of ligase chain reaction is based in part on the ligation of two adjacent synthetic20 oligonucleotide primers which uniquely hybridize to one strand of the target DNA or RNA. If the target is present, the two oligonucleotides can be covalently linked by ligase. A second pair of primers, almost entirely complementary to the first pair of primers is also provided in a ligase chain reaction. In a ligase chain reaction, the template and the four primers are placed 25 into a thermocycler with thermostable ligase. As the temperature is raised and lowered, oligonucleotides are renatured adjacent to each other on the template and ligated. The ligated product of one reaction serves as the template for a subsequent round of ligation. The presence of target is manifested as a DNA
CA 02221467 1997-ll-18 W O96/36731 ~ PCTIUS96/06527 fragment with a length equal to the sum of the two adjacent oligonucleotides.
Additional PCR variations include in situ PCR and imm-mo-pCR amplification which utilizes nucleic acid fr~gmtontc coupled to pathogen-specific antibodies to increase detection sensitivity. Alternatively, nucleic acids can be analyzed after 5 purification using, for example, DNA or RNA polymerases, PCR or another amplification technique. PCR analysis of RNA, or RT-PCR, involves reverse transcription of RNA, such as mRNA sequences, into cDNA copies. These target cDNA sequer~r~s are hybridi_ed to primers which ampli~y the nucleic acid using PCR amplification.
Although high level amplification may be possible, it may not always be n~PC~, y or even desired when, for example, tLc sequence amplified is likely to mutate or otherwise be altered during the amplification process. Insuch cases, PCR can be limited to just a few rounds of amplification or avoided altogether and sequence replicated using more conventional nucleic acid 15 polymerases.
The sequence of the nucleic acid including the target sequence will be .1~ P~ by the sequence of the nucleic acid obtained from the sample.
However, synthetic seq~lenr~s may be added or the entire nucleic acid may be synth~tir~lly synth~si7~1 As such, nucleic acids may comprise any conlbi,~lion 20 of purines or pyrimi~lines, mo~ifir~tions or derivatives of purines or pyrimidines, or other ch~mir~l moieties which can be hybridi_ed specifically or non-specifically to a nucleic acid sequence. For example, neutral bases, those bases which non-specifically hybridi_e to most any other base, such as inosine or modifications or derivatives of inosine, can be incorporated. In addition, 25 incorporation of residues such as thiolated bases, boronated bases, polyamides and peptide nucleic acids can produce sequences which are resistant to Cll 'ylllaLiC
degradation.
Sequences of the nucleic acid, including the target scquence, may encode protein or be entirely non-coding sequences such as structural sequences or seqllrnr~c which regulate expression. Structural sequences include ribosomal RNA and telomeres. Controlling sequences include promoter sequences, S enh~nr~rs, 5'- and 3'-untr~n~l~ted sequences and sequences tl~at function outside of expression such as ribozymes. Identific~tion of variations within such sequences can be important in determining Llc:allllcnt regimr-nts, such as in ideneifying repeat numbers, in d~lellnil~illg molecular structure and in generating relationships. For example, target sequences within the nucleic acid may be 10 sequences which are specific to a particular species or strain of organism such as a ba~;Leliul,l, virus, parasite or fungus, or the sequence of a tr~ncl~t~d oruntr~n~l~ted portion of a eukaryotic or prokaryotic gene. Identifir~tion of suchsequences can be used to detect and often identify the org~ni~m Alternatively, the target sequence may c~ e a homologous seq~l~nr~ such as inosine, uracil 15 (U) or deoxyuracil (dU), when only the length of the target sequence is to be determin Nucleic acids are hybridized to an array of probes by any ~lumbel of techniques known to those of ordinary skill in the art. For example, hybridizations may be performed in a buffered salt solutions such as SSC (3M
20 NaCl, 0.3 M Na Citrate, pH 7.0), or SSPE (3M NaCl, 0.2 M Na Phosphate, 0.0~ M EDTA, pH 7.4). Other solutions can be utilized where melting temperature of the double helix is independent of base composition and dependent only on length. Solutions which have this property include solvents cont~ining quaternary alkylammonium salts such as solutions of tetramethyl-25 ammonium chloride or tetraethylammonium chloride. In quaternaryalkylammonium solutions the bonding strength of ~T base pairs and GC base pairs are approximately the same.
Probes of the array each comprise regions which are compl~ to one or more portions of the nucleic acid. Preferably, probes comprise 5'-region and 3'-regions which are complementary to portions of the nucleic acid and an internal variable region. The variable region can vary in 5 sequence and/or length and, preferably, one of the variable region sequences of the array is complt-m~ont~ry to or will otherwise completely hybridize to the target seq lenr~. Variations in probe sequence will prevent certain of the probes from fully hybridizing to the nucleic acid cont~ining the target sequence. Theseheteroduplexed probes, conr~ining an unhybridized portion in either the probe 10 or the nucleic acid, are susceptible to digestion using a single-strand specific nuclease.
Probes and nucleic acids may be i(lentir~lly or dirr~ Lially labeled with ~letr~t~hle labels. Detectable labels include radio-isotopes such as '25I, 35S, 32p or 3H, stable-isotope or ch~mir~l moieties such as a fluorescent,15 l~ i..rsc~ or chemilllmint~scrnt compounds. Additional labels which may be used include chromogenic chemicals, metals, coupling agents such as biotin/streptavidin or avidin, mass modifying moieties, m~gn~tic agents or chrmir~ et~o~t~hle by nuclear m~nPtir rrson~nre or electron spin resonance.
Labels may be incorporated enzyrn~tir~lly, for example, during gt;ll~.alion of the 20 nucleic acid or by rh-omic~l m~lifir-~tion of the final structure. Specifically useful labeling compounds are those which do not ..~ relc with the polymerase reaction such as rhodamine, fluorescein, dansyl chloride, coumarin, digoxin, fluoresc~minP and derivatives and modifications of these compounds.
Probes or target nucleic acids may also be fixed to a solid support 25 or free in solution. When free in solution, hybridization may be in an ordered fashion such as in well separated wells of a microtiter dish or multi-well chip,or together in a single well or small number of wells. In this fashion, batch analysis of hybrids can be performed sequentially to minimi7~ the number of ~ probes needed to identify an unknown target sequence. ~Iternatively, probes can be hybridized to nucleic acids in an ordered fashion such that individual hybridization events can be accurately scored. Useful solid supports include plastics, glasses, ceramics, metals, resins, gels, membranes, chips such as 5 hybridization chips, and combinations of these materials and structures.
This hybridized array, either fixed or free in solution, is digested with a single-strand specific nuclease to cleave single stranded regions such asheteroduplexes and terminal extensions. Nucleases suitable for digestion of hybridized probes include those nuclease which plcfe~cllLially cleave single-10 stranded nucleic acids. P~efe--cd nucleases include the endonucleases such asSl mlrle~e, mung-bean nllc le~ce, ribom-cle~e A and ribon--rle~e T1. Nucleic acids or probes which generate l~ single strands can be digested with exon--rle~es such as the T4 and T7 phage nucleases. When desired, tre?,tm~nt with excess mlrle~e can be directed to produce double-stranded cleavage by 15 eYt~n~ling the nick to a gap and thereby creating a single-stranded region on the opposite strand. Such double-stranded cuts can be useful in procedures where probes are fr~gmrnt~
Nicked hybrids can be labeled using tennin~l deo~L al~rc-~se or another suitable nucleic acid modifying enzyme, and precursor dNTPs or ddNTP
20 d~tert~bly labeled with a radio isotope, stable-isotope or chemical moiety such as a~ fluorescent, l lminrscent or ch~-mihlmin~scent moiety. Additional labels which may be incorporated include chromogenic ch~mir~l~, metals, coupling agents such as biotin/streptavidin or avidin, mass modifying moieties, m~gn.otiragents or ch.omic~ etect~ble by nuclear m~gntotic resonance or electron spin 25 resonance.
Digested hybridized probes are then contacted with a nucleic acid polymerase to extend nicked strands and thereby displace one strand of the heteroduplex. Polymerases which can be used for elongation include any polymerase which can elongate a template after a nick. Most DNA polymerase of most org~nicmc are suitable for the practice of this invention. Examples of suitable polymerase include human DNA polymerase I, II, and m, E. coli DNA
polymerase I, II, and m, T7, T3, and SP6 polymerase, thermostable DNA
5 polymerase, sequenase, and amplitaq polymerase.
An~her embodiment of this invention is directed to a method to measure the length of a target sequence. Probes constructed for length mea~u~cll~ents preferably comprise neutral bases such as inosine residues flanked by two constant region sequences. An advantage of neutral bases in that a 10 knowledge of the target sequence is not required. Neutral base forms stable base pairs with all four conventional bases and the strength of the paring is approximately equal in each case. With the use of a neutral base, the assay willbe sensitive only to the length, but not the sequence of the target.
Another embodirnent of the invention is directed to a method for 15 detecting the number of repeat sequences in a target nucleic acid. A target sequence may be from a natural source or a synthetic source. Natural sources of target sequence may include DNA, and RNA from an org~ni~m The nucleic acid may be from seq l~onrPs which encodes a protein, such as exons and mRNA.
The nucleic acid may also be from structural and from non-coding sequences 20 such as ribosomal RNA, and telomeres. Genes which comprise repeated sequences, such as human TFIID and human DNA polyrnerase II largest subunit, have internal trinucleotide repeats which encodes for strings of homopeptides whose length varies between individuals. Non coding repeat sequences include the repeating DNA and telomeric sequences. Synthetic sources of nucleic acids 25 may be from a laboratory reaction, a nucleic acid synthesis machine. Additional sources of nucleic acids may be from nucleic acids added to industrial and consumer goods.
-W O96/36731 PCTrUS96/06527 To determine the number of repeats in a target sequence, the target sequence is hybridized to a plurality of probes, each cont~ining none, one or more than one repeat. Where the number of repeats in the target do not correspond to the number of repeats in the probe, one or more single stranded S loop can be present on the target-probe hybrid. Single stranded loops are onlyabsent in the hybrid with a perfect match. Perfect m~tch~s co..~ hybrids of nucleic acid target to probes with the same number of repeats. Single strand nuclease treatrn~qnt after hybridization will digest all the single stranded loops leaving nicked hybrids and un-nicked hybrids. Polymerase tre~tmrnt after 10 digestion elongates and displaces strands of all nicked hybrids. Hybrids with a perfect match and without nicks will be the only hybrids not affected by polymerase. By m~ o. ill~, the polymerase reaction, the hybrid with the perfect match can be identified and the number of repeats in the target can be detel...i..~ cl.
The polymerase reaction can be monitored by a llu~ el of mrthod~. The polymerase elongation reaction may be ~,r~ ed in the presence of nucleotide triph~ sph~ttos with a detect~hle moiety. On ~letPct~hle moiety is a radio-label such as 32p or 35S on the a-phosph~tr. All the hybrids with an incorrect number of repeated se~enr-e will be labelled while the hybrid with 20 equal number of repeats will remain unlabeled. Thus, the assay allows for theprecise identifir~tion of the numher of bases or the number of repeat seq~rnres in a target sequence. As such, these methods are faster and more sensitive than methods currently available.
Another embodimem of the invention is directed to a method for 25 screening a patient suspected of having a genetic disorder. A sample of tissue is obtained such as a sample of tissue or bodily fluid, and nucleic acid PCR
amplified, purified or cloned. The target nucleic acid sequence is hybridized toan array of probes, nuclease and polymerase treated and the presence or absence CA 02221467 1997-ll-18 W O96/36731 PCTrUS96/06527 of the genetic defect ~l~tect~d. Disorders which can be ~lettocte(l include, forexample, lllyotonic dystrophy, ~llntington~ s disease, Kennedy disease and Fragile X ~ylldlulllc. Patients may be any m~nnm~l such as a human. Patient s~mplç~ may be collected and pooled to reduce the number of tests which need to be performed to identify a positive carrier, or sequentially analyzed againsta variety of different probe arrays to further limit the number of tests and probes needed.
Another embodiment of the invention is directed to arrays of probes wherein each probe comprises a constant 5'-region, a constant 3'-region 10 and a variable internal region wherein the variable region comprises one or more repeat sequences. The repeat sequence comprises heterologous or homologous sequences which are variable in length or base sequence. Sequences contain purine or pyrimi~lin~ bases or neutral bases such as inosine. Either the nucleicacids or the probes of the array may be labeled with a detect~hle label or-fixed15 to a solid support. Arrays may be spatially ordered by structure or sequence with the seq~l~n~s of the probes known or ~ hle. Probes may be single-stranded or partly single-str~nrl~ and partly double-stranded. Probes may also be labeled with dett~ct~ble labels. Arrays may comprise between about 10 to about 10,000 dirr~lelll probes, preferable between about 50 to 5000 dirrelclll 20 probes, or more or less as required.
The following e~.illlents are offered to illustrate embo lim~n of the invention, and should not be viewed as limhing the scope of the invention.
Fxamples Example 1 Oli~onucleotide Syn~hesis. Purification. and Characl~ tion.
Synthetic oligonucleotides comprising the following sequence were synthesized using an oligonucleotide synthesizer (Operon Technologies, Inc.). The sequences of the oligonucleotides are as follows:
W O96/36731 PCTrUS96/06~27 Tl(78 mP~
5'-CCAGATCTGA TGCGTCGGAT CATCCAGCAG CAGCAGCAGC
AGCAGCAGTC ACGCTAACCG AATCCCTGGT CAGATCTT-3' (SEQID NO 1) T2(78 mer) S'-AAGATCTGAC CAGGGATTCG GTTAGCGTGA CTGCTGCTGC
TGCTGCTGCT GCTGGATGAT CCGACGCATC AGATCTGG-3' (SEQID NO 2) CTG6(72 rn~r) S'-AAGATCTGAC CAGGGATTCG GTTAGCGTGA CTGCTGCTGC
TGCTGCTGGA TGATCCGACG CATCAGATCT GG-3' (SEQID NO3) Oligonucleotides T1 and T2 were purified by polyacrylamide gel electrophoresis, while CTG6 was purified by using high pelrcllllaLce liquid 15 cl~,o~a~ography. The concentration of each stock solution was ~leterrnin~l by absorption at 260 nm.
Tl,T2 and CTG6 contain 8 GAC repeats, 8 CTG repeats, 6 CTG
repeats, respectively. The GAC repeats are located 30 bases from the 5' end and 24 from the 3' end. The CTG repeats are located 24 from the 5' end and 20 30 from the 3' end.
Example 2 Deternlin~tion of S1 Nuclease Specificity ~n-l F.fficienry.
S1 mlcle~e specificity and effirien~y was monitored using 5' radio-labeled oligonucleotides. Briefly, 3.5 ,uM of oligonucleotide was placed in kinase buffer (70 rnM Tris-HCl, pH 7.6,10 rnM MgCI2, 5 mM dithiothreitol) 25 cont~inin~ 6.4 pM 3~P-ATP (specific activity of60 Ci/mrnole). End labeling was initi~t~l by the addition of 0.35 unit/pmole oligo T4 polynucleotide kirlase(New Fn~l~n~l Biolabs; Beverly, MA). Labeling continnecl for 45 mimlt~s at 37~C. Labeled oligonucleotides were separated from unincorporated 32P-ATP
with a CHROMA-SPINTM+TE 10 columns (Clonetech).
CA 02221467 1997-ll-lX
W O96/36731 PCTrUS96/06527 Heteroduplexes were g~ t~d by ~nnr~ling 1 ,uM of 32P-labeled oligonucleotide T1 to an equal molar amount of T2 or CT66 in a 50 pL volume of 100 mM Tris-HCI, pH 8.0 (Figure 1). Oligonucleotides were heated to 96~C
for four ...i....~ and gradually cooled to 30~C over two hours to ensure specific S ~nn-o~ling.
The specificity of S1 as a function of enzyme concentration was tested using T1-T2 and Tl-CTG6 heteroduplexes labeled as Hl and H2, respectively, in Figure 1. Briefly, 0.1-1.0 unit/picomole of S1 nuclease (Promega; Madison, WI) was added to the heteroduplexes in a solution of 200 10 mM NaCI, 50 mM sodium acetate, pH 4.5, 1 mM ZnSO4, 0.5% glycerol.
Nuclease digestion was ~lr~ ed at temperatures of abou. 0~C, about 24~C and about 37~C. The L~lll~lalul~S of the solutions were equilibrated to the reactiontelllpelàture before the addition of enzyme. After a reaction period of 60 minlltes, further digestion was stopped by the addition of EDTA to a final 15 concentration of 12 mM. .Sch~m~tirs of the expected reaction products are shown in Figure 1 C, and D. Each reaction product was analyzed by native 12% polyacrylamide gel electrophoresis. R~cllltin~ gels were autoradiographed and are depicted in Figures 2A and 2B. Figure 2A depicts an autoradiograph of the reaction product of the perfect match heteroduplex T1-T2. T ~ne 1 is a minus20 S1 control. Lanes 2-5 contain increasing collcellLIalions of Sl (0.2, 0.5, 0.8, 1.0 units per picomole oligo) all incubated at 0~C. Lanes 6-9 contain identical concentrations, but were inr~h~ted at room telll~elalule, and lanes 10-13 were incubated at 37~C. Although at higher temperatures S1 cut the end label off of the duplex, no other cutting was seen. Lane 0 contains size standard 25 (~X174/HinfI digest).
Figure 2B is an autoradiograph of the reaction product of the mi~m~tched heteroduplex T1-CTG6. Lanes 1-4 contained increasing concentrations of Sl (as above), all inr~lb~ted at 0~C. Lanes 5-8 follow the W O96/36731 PCTrUS96/06527 same pattern of S1 concentration, but were in~lb~tP~ at room temp~rature, while lanes 9-12 were inr~lbatP~ at 37~C. Both lanes 13 and 14 contain T1-CTG6 complex without any Sl mlclP~e. The top band in each lane (band A) m~tchPs with the T1-CTG6 control and is just the uncut loop structure. The second band 5 (band B) is the nicked loop, while band C appears to be a nicked loop that hasbeen partially digested. Lane D is very faint, but may contain completely digested loop, leaving a nicked duplex DNA. T ~ne 15 contains a size standard.
At 0~C, greater than about 60% of the 6 base loops gel~eratcd by the mi~m~tchPA repeats in the Tl-CTG6 hybrid complex were cut by S1 nuclease 10 at a concentration of 0.6 units per picomole (Figure 2). The presence of multiple bands was most likely due to S1 nn~le~e cleaving the loop structure and thereby degrading several unpaired nucleotides. It also appears that S1 nuclease cut several u~ail~d nucleotides rather than just one, since distinct bands appeared at separations of more than one base pair. In contras~, no 15 cleavage was seen with the pc,re~;lly m~t~hP~l Tl-T2 hybrid complex.
At higher (~ l cs7 less of the label ap~cd in each lane of both the m~tchP~l and ...~ hPCl samples. This was most likely due to Sl nuclease cleaving the b-call~illg ends of duplex DNA as single-stranded structures were formed. This problem was not seen in samples inrnb~tPd at 0~C
20 because the extent to which the DNA ends could breath was reduced. These experiments ~lemo~ aled that S1 nllcle~ce cleaved the hybrid cont~ining a micm~tch at the location of the mi~m~trh Example 3 T ~helir~ ~n-l Strand Displacement.
An enh~n~e~l method to discriminate between the m~tfhP-l and 25 mi~m~tchPA oligonucleotides was e~minPA . Labeling and strand displ~- emPnt reactions were tested with templates con~icting of unlabeled T1-T2 and T1-CTG6 heteroduplexes. Digestion of these duplexes was performed with 0.6 units of S1 nuclease per picomole of oligonucleotide at 0~C. Reactions were W O96/36731 PCTrUS96/06527 ter~i~L1ated and the products purified with a spin column (CHROMA-SPI~M+TE 10). S1 nllcle~ce was inactivated after column purification of the oligonucleotide because of the removal of ZnSO4.
The ex~. ~l~..L~l scheme and the expected results are represented 5 in Figure 3. The expected digestion products of the mi.cm~tch~l heteroduplex is represented as Al while the expected digestion product of the perfect match heteroduplex is represented as A2. The expected reaction product after polymerase treat~lnent is shown as B1 and B2, respectively.
Labeling of the Sl digested heteroduplexes were performed for 10 15 ~ s at room t~lll~ldtU~ with the Klenow fragment of DNA polymerase I. Briefly, 0.08 units per picomole of enzyme was added in a reaction buffer of 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 0.001% gelatine, 30 pM of each dNTPs, and 32P-labeled dCTP (specific activity of 1.74 Ci/mmole) in a volume of 50 pl. The reaction was stopped by addition of sodium dc~decyl 15 sulfate (SDS) to a final concentration of 0.5%.
The product of the labeling reaction was analyzed by ac.-ylamide gel electrophoresis and autoradiography. A copy of the autoradiograph is shown in Figure 4. Lane 0 is a molecular weight m~rker. Lane 1 and Lane 2 represents S1 digested and polymerase treated micm~t(~.h.--l heteroduplex 20 elongated in the presence (lane 1) and absence (lane 2) of radioactive nucleotide triphosphates. Lane 3 and lane 4 represents S1 digested a~d polymerase treated perfect match heteroduplex elongated in the presence (lane 3) and absence (lane 4) of radioactive nucleotide triphosphates.
Incorporation of 32P-labeled of dCMP in the S l-cleaved, 25 mi.cm~s~hed hybrid (Tl-CTG6) by Klenow fr~gm~nt yielded a strong signal at the position expected if the S1 cleavage occurred at the site of the micm~s~.h (Figure 4). Omy a very weak signal could be ~let~-ct~ for t'ne perfectly m~t~h~lhybrid (Tl-T2), and this signal was not localized into any distinct bands. Some CA 02221467 1997-ll-18 W O96/36731 PCTrUS96/06527 non-specific labeling of the perfectly matched hybrid, as ~vell ac the T1-CTG6 complex may have arisen from the t~?n~l.on(~y for Sl nuclease to introduce nicksinto double-stranded DNA. However, the loop-cutting activity of Sl nuclease is much stronger than its ability to introduce nicks into perfectly m~trhtocl 5 double-stranded DNA, which is demonstrated in these experiments.
Example 4 Detection of a Repeated Genomic Sequence.
A single-stranded nucleic acid co~ fl~ g an internal target repeat sequence is generated from genomic DNA for analysis. A sch~ tic of the strategy is shown in Figure 5. Briefly, one 5'-biotinylated oligonucleotide 10 primer and one non-biotinylated primer is produced using an oligonucleotide ~y~ r. The ~ el:i flank a region of genomic DNA cont~ining a variable number of repeated nucleotides. A polymerase chain reaction is performed using the two primers and genomic DNA as template (Figure SA). Double stranded reaction product is purified from unincorporated nucleotide 15 triphosphates by a size eY~ cion column. The purified PCR product is de~ ued in 8M urea and the biotinylated strand removed. The non-biotinylated strand is labeled at the 3' end with a fluolesc~ill and used as the target nucleic acid.
A plurality of probes, each cont~ining 5' and a 3' sequence 20 comple.~ to the target nucleic acid and from 10 to 109 internal repeats are synthPci7~oA on an oligonucleotide synth~--ci7~r. Probes of 80 bases or shorter are synth~ci7~l and used directly. Probes greater than 80 bases in size are synthesized as fr~mentc and ligated together. After generation, probes are labeled at the 3' ~ lc with rho~minP. All the probes are synth~ci7e~ with 25 a 5' biotin and these biotinylated probes are ~tt~ch~d to the bottom of a plate coated with immobilized streptavidin. Probes are attached along a 10xlO array and ordered according to size (Figure SB).
WO96/36731 PCT/US96/06~27 Target nucleic acid is hybridized to the probe array (Figure SC) and digested with S1 nuclease (Figure SD). DNA polymerase is added to the array and elongation and strand displ~r~m~-nt is allowed to occur (Figure SE) until completion (Figure SF). When the probe contains more internal repeats S than the target, the rh~l~mine label will be lost in the strand displ~em~nt and the r~-slllt~nt proa-lct will be red. Similarly, when the target contains more internal repeats than the probe, the fluorescein label will be lost and the product will be green. When the probe and the target both contain the same number of repeats, both rhodamine and fluolcsceill will remain and the reslllt~nr color will 10 be yellow.
After strand displacement the array is inspected visually. The result is displayed in Figure 6. All the probes are yellow before strand displ~ment (Figure 6A). After S1 cutting and strand displ~f~m.ont, the probes with fewer repeats than the target is red and the probe with more repeats is lS green. The probe with the sarne number of repeat is yellow. The results of expe~ c~ clrolllled with the same probe array but with target DNA
comprising 88, 55, and 17 repeats are shown in Figure 6B. This experiment demonstrates how a colormetric assay may be performed to delcl~ e the nurnber of repeats in a target sequence.
20 Example S Detection of Repeated Seque~o from Myotonic Dystrophy Patient.
To (~ the extent of expansion of trinucleotide repeat in a myotonic patient, a S ml sample of blood is drawn from the patient for ~n~lysis.Whole cell DNA is isolated from the blood and a DNA, comprising a region of 25 trinucleotide repeats, implicated as a cause for myotonic dystrophy disorder, is amplified and isolated by polymerase chain reaction. Polymerase chain reaction products are denatured and one of the DNA strands used as the nucleic acid cont~ining the target sequence to be detected.
CA 0222l467 l997-ll-l8 W O96/36731 PCTrUS96/06527 ~ An oligonucleotide synthesizer is used to generate a set of oligonucleotide probes. Each probe in the set has a 20 base-pair 5' sequence anda 20 base-pair 3' sequence compleTnPnt~ry to the sequence fl~nking the trinucleotide repeat region. In addition, each probe in the set has an internal 5 trinllrlPotide repeat between the 5' and 3' sequence. A series of 20 probes are synthesized cont~ining from 1 to 20 trinucleotide repeats.
Three picomoles of each probe, a total of 60 picomoles, is hybridized to 200 pmoles of the amplified target nucleic acid. Briefly, the probes and the targets are heated in 100 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 to 96~C for four minllt~Ps and cooled gradually to 30~C over two hours to ensure specific ~nnP~Iing to form heteroduplex with mi.~m~tc~ s and perfect m~t llPs.
Heteroduplexes are treated with 0.3 unit per picomole of Sl ml~le~e at 0~C for S mimltes. The reaction is stopped by chromatography of the reaction ~ we through a spin column.
Polymerase tre~tmPnt of the Sl digested heteroduplexes is performed for 15 ..ii....~es at room l~u~ d~ul~ with the Klenow fragment of DNA polymerase I. Briefly, 0.08 units of enzyme is added per picomole DNA
in a reaction buffer of 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 0.001% gelatine, 30 pM of each dNTPs. The reaction is stopped by ~ lifi~n of sodium dodecyl sulfate (SDS) to a final collcel.l.dlion of 0.5%.
The product of this reaction is analyzed on a delldlul ing sequencing gel with the set of DNA probes as a molecular weight marker. After electrophoresis, the gel is treated with water for 30 minutes to remove the ureaand stained with SBYR or FBIR. Bands are ~ tected upon exposure to ultraviolet light. The largest product observed is a 61 base band corresponding to 7 trinucleotide repeats.
Other embo lim~ontc and uses of the invention will be apl)alelll to those skilled in the art from consideration of the specification and practice of t;le W O96/36731 PCTrUS96/06527 invention disclosed herein. All U.S. Patents cited herein are specifically incorporated by lt;r~lellce. The specification and examples should be consideredexemplary only with the true scope and spirit of the invention in-1ir~te~1 by the following claims.
s
Ri~hts In The Invention This invention was made with United States Gov~ support 5 under grant llulllbel DE-FG02-93ER61609, awarded by the United States Department of Energy, and grant number AIBS2154, awarded by the United States Depallmenl of the Army, and the United States Go-vellllllen~ has certain rights in the invention.
BackFround of the Invention 10 1. Field of the Invention This invention relates to methods for the detection of repeated and other j~lentifi~hle nucleic acid sequences. The invention also relates to methods for identifying and mapping specific nucleic acid sequences in complex backgrounds.
15 2. Descli~ion of the Background Historically, the ~ gn~ si~ of genetic disease has depended on the irl.s~.lir~lion of abnormal gene products or their clinical effects such as ~n-omi~, mental retardation and certain sch-~hrenia. Through direct analysis of the genome, it is possible to identify genetic mllt~ti~ns and offer tre~tm~nt before20 the ~ ir~ lion of ~ylll~OlllS. Genetic analyses ~elÇoll,led today range from gross analysis such as karyotyping to the analysis of individual base pairs by sequencing. Although a great deal of progress has been made, nucleic acid sequencing is still too labor hllel~ive and e~-~ensive for everyday diagnosis beyond the experimental m~ 1 research laboratory.
Many genetic defects such as Burkett's Iymphoma and some sickle cell anemia and th~ semi~ mutations are ~letect~hle without the use of sequencing. Such techniques include restriction fragment length polymorphism Q (RFLP) and chromosome karyotyping. However, general applicability of these methods is limited as most genetic defects are more modest and do not alter 30 restriction sites or cause chromosome rearrangements. Polymerase chain WO96/36731 PCTrUS96/06S27 reaction and ligase chain reaction can increase the sensitivity of many detection methods and detect single base pair changes in nucleic acid. However, if the mnt~tion involves repeated sequences, the degeneracy of the repeated sequence makes even PCR and LCR detections unreliable.
S Dinucleotide and trinucleotide repeat sequences are increasingly beco~ lg important in genetic analysis. These repeats are both polymorphic and widespread in the human genome and offer a convenient means for locating genes associated with particular phenotypes (M.S. Wehnert et al. Nuc. Acids Res. 22:1701-4, 1994; G. Benson et al., Nuc. Acids Res. 22:4828-36, 1994).
Trinucleotide repeat expansion mutations have been ic1entifie~1 in at least four human genetic ~ e~e,s (C.T. Caskey et al., Sci. 256:784-89, 1992). Each are caused by mutational m.-ch~ni.~m~ whereby normally polymorphic exonic trinucleotide repeats expand beyond the normal size range and alter gene eA~l~,s~ion, mRNA stability or gain certain functions. In FragileX ~yll~ e (FraX; D.L. Nelson et al., Nature Genetics 4:107-108, 1993), the second most col...l~ll genetic form of mental retardation, and also in myotonic dy~ ophy (MD; D.J. Brook et al., Cell 68:799-808, 1992), the repeat e~p~n~ion can be quite large resl-lting n thousands of triplets. In spinal and bulbar mllecul~r atrophy ~SBMA or K~nnPAy disease) and ~llntington's Disease (HD), 20 the exp~n~ion may only consist of twice the normal compliment of repeats.
The genetic element expanded in Fragile X is a triplet called FMR-l. This sequence, CGG, is highly polymorphic in the general population ranging from between about 6 to about 42 triplets per person. Unaffected family members can contain up to 50 repeats. Between 50 and 200, individuals are 25 considered to be pre-mutation. Expansions of several thousand are known to occur in affected patients.
W O96/36731 PCTrUS96/06527 Myotonic dystrophy is an autosomal dominant disorder characteri_ed by muscle we~kness and is the single-most common form of adult ~ onset. The gene responsible, DM-1 has been i~entified There are many methods for ~letecting differences in repeat 5 num'oer. Conventional analyses involve electrophoretic fracti~nation steps. Such steps are seriously limiting in terrns of time and expense and lack the se~ iviLy for detecting short deletions in long sequences (M.B. White et al., Genomics 5:301-6, 1992). Ch~-mic~l detection and cleavage of mi~m~tches, though effective, generally relies on the use of dangerous compounds (P.M. Smooker 10 et al., Mutant. Res. 288:65-77, 1993). The advent of efficient coupling of DNA
to solid surfaces as well as progress in effective flc lcscell~ labeling and ~lett-~tion have paved the way for the development of assays able to detel~ le the length of these dinucleotide and trinucleotide repeats quickly and accurately.
Slllnl l l~ of the Invention The invention overcomes the problems and disadvantages associated with current strategies and designs and provides novel methods for the detection and id~ i ri~ ion of nucleic acid sequences and novel arrays whichcan be ~tili7PA with these methods.
One emb~lim~ont of the invention is directed to methods for 20 ~eL~ g a target seqllen~e within a nucleic acid. The nucleic acid is hybridi_ed to an array of probes wherein each probe com~lises a 5'-region complemlont~ry to the nucleic acid, a 3'-region complem~nt~ry to the nucleic acid, and an internal variable region. The hybridi_ed array is digested with a single-strand specific nuclease and treated with a nucleic acid polymerase. The target 25 sequence may vary in length or sequence, for exarnple, comprising a pluralityof short repeat sequences or a homologous sequence of bases of variable lengths.The sequence and length of the target can be identified by hybridization to a specific probe and resistance to the single-strand specif1c nuclease.
W O96/36731 PCTrUS96/06527 Another embodiment of the invention is directed to -nethods for d~ ing the length of a target sequence within a nucleic acid. A nucleic acid is hybridized to an array of probes wherein each probe comprises a 5'-region complementary to the nucleic acid, a 3'-region complementary to the nucleic S acid, and an internal variable region. The hybridized array is digested with asingle-strand specific nuclease and treated with a nucleic acid polymerase. The nucleic acid may be a PCR product, such as an amplified nucleic acid sequenre, or a DNA or RNA macromolecule purified, if n~-cess~ry, directly from a biological sample. The internal variable region may comprises a homologous 10 sequence of bases such as a sequence inosine residues which non-specifically hybridize to nucleic acids. Hybridized probes resistant to nllrl~e digestion will be the same length as the target sequence.
Another embc~lim~ont of the invention is directed to methods for drL~ .1l;. .;. ~ the number of repeat seq~onres within a nucleic acid. The nucleic 15 acid is hybridized to an array of probes wherein each probe comprises a 5'-region complem~o-nf~ry to the nucleic acid, a 3'-region complem~ont~ry to the nucleic acid, and an internal region which contains one or more repeat sequences. The hybridized array is digested with a single-strand specific mlrlto~e and treated with a nucleic acid polymerase. Hybridized probes rt;:j~L~lL
20 to the nuclease digestion contain the same number of repeats as the target sequence.
Another embodiment of the invention is directed to methods for screening a patient suspected of having a genetic disorder. A tissue sample is obtained from the patient and a nucleic acid sequence obtained by, for example, 25 PCR amplification or direct purification of a target sequence. The nucleic acid is hybridized to an array of probes wherein each probe comprises a 5'-region and a 3'-region, each complementary to the nucleic acid and a variable internal region. The hybridized array is digested with a single-strand specific nuclease and treated with a nucleic acid polymerase. Hybridized ~robes resistant to nuclease digestion will contain a specific number of repeat sequences. The plesellce or absence of the genetic disorder can be deterrnined from the ~ be of repeat sequences which are present.
Another embodiment of the invention is directed to arrays of probes wherein each probe comprises a con~t~nt 5'-region, a constant 3'-region and a variable internal region wherein the variable region comprises one or morerepeat sequences. The repeat sequence comprises heterologous or homologous sequences which are variable in length or base sequence. Sequences contain purine or pyrimidine bases or neutral bases such as inosine. Either the nucleic acids or the probes of the array may be labeled with a d~tect~ble label or fixedto a solid support.
Other emb~lim~onf~ and advantages of the invention are set forth, in part, in the description which follows and, in part, will be obvious from this description and may be lc~nltoA from the practice of the invention.
Descri~tion of th~ Drawin*.~
Figure 1 Sch~m~ti~ of the reaction strategy.
Figure 2 Results of micm~tl~h cleavage with S1 nuclease.
Figure 3 Labeling of S1 cleavage products with radio-labeled nucleotides.
Figure 4 DNA polymerase radiolabeling of Sl cleaved m~tch~l and mi~m~trh~cl substrates.
Figure S .Sch.om~ for detection of mi~m~tch~s using anchored single-stranded oligonucleotide probes.
Figure 6 Two dimensional array for the detection of between 10 to 109 repeats.
Pescription of the Invention As embodied and broadly described herein, the present invention is directed to methods for the detection and i-lPntifi~tion of target sequences by size or base sequence and to arrays of nucleic acid probes which can be utilizedwith these methods.
Nucleic acid screening is widely utilized to detect and identify nucleic acids. The presence or absence of these specific nucleic acids, as S identified by their sequences, can often be considered as evidence of disorders such as infections, neoplasms and genetic diseases. Although there are a wide variety of methods ~;~nlenlly available, sequence detection is generally a slow and expensive proposition requiring costly supplies and the skills of highly trained individuals.
It has been discovered that by combining certain microchemical tools such as nucleic acid probes, nucleic acid hybridization and enzymatic cleavage of heteroduplexed hybrids, procedures can be designed to detect specific target seqllenrPc. Char~cte-ri~tir sequences such as occurs in variations between strains of microorg~ni~m~ and between numbers of repeat sequences can 15 be rapidly and accurately AetectrA and iArntifird Nucleic acids co..l;.i.-i..g these target se~ ellces can be hybridized to oligonucleotide probes that contain seclllenre variations such as a differentrepeat lengths. Loop structures formed by ...ic..~ r,r~ repeats can be cleaved by inrllb~tion with a mlrle~e to geu~,ldle nicked double strands. These nicks 20 are recognized by a nucleic acid polymerase which breaks down or displace one of the strands. Analysis of the products using, for example, dirr~ ial labeling, reveals the nature of the ...i';...~lch as well as the length of the perfectly m~tc'nt-A repeats. As reactions can be co. AIlcte~ in situ and all under the same conditions, process steps can be easily ~uL~ll~d. Many assays could be run in 25 parallel allowing for rapid analysis of target sequence from a variety of sources.
One embodiment of the invention is directed to a method for A~tecting a target sequence within a nucleic acid. Nucleic acids cont~ining target sequences to be ~Tetect~A can be obtained directly or indirectly from natural or W O96/36731 PCTrUS96/06527 ~yllLll~Lic sources. Synthetic sources include sequences chemically synthesized such as oligonucleotides or sequences of PNA. Natural sources of nucleic acid sequences include samples of bodily tissues or fluids obtained from a patient, samples from the environment such as a biomass, soil or body of water. Nucleic acids directly obtained from such sources can be purified, if npcess~ry~ by techniques such as centrifugation, chromatography, chemical extraction, i~lion or other techniques or combinations of techniques known to those of ordinary skill in the art. As sequence information is easily transcribed or replicated, the nucleic acid may be either RNA or DNA and may exist in either the sense or anti-sense orientation.
Nucleic acids are preferably single-stranded, but may be partially single-stranded and partially double-str~n-l~ Single-stranded regions hybridize to probe sequences and double-strand regions can contain recognitions sites for restriction enzymes or other nucleic acid modifying enzymes sites, or used to ch~ lly couple ~let~t~hle labels. If n~cecc~ry, single-stranded nucleic acids can easily be ~r~aLed from target sequences by a number of methods. The strands of most double helixes, once denatured by ~lc~ with 8M urea, low or high pH or 95~C heat, can be s~alal~d by, for example, del~Luli lg electrophoresis. ~lt~rn~tively, polymerase chain reaction using one or an excessof one primer may be performed using the target seq~enre as a template causing the product to consist mainly of one strand. Elongation products formed, for example, using a biotinylated primer can be isolated with a streptavidin column.mRNA, or single stranded cDNA may also be isolated and used as a single stranded target.
The nucleic acid cont~ining the target sequence is preferably generated as a polymerase chain reaction (PCR) product. The basic PCR
process is described in U.S. Patent No. 4,683,195. Variations of the PCR
process are described in U.S. Patent Nos. 5,043,272, 5,057,410 and 5,106,727.
CA 02221467 1997-ll-18 W 096/36731 PCTtUS96tO6527 As a PCR product, the nucleic acid will possess both 5' and 3' terminal sequences which are i(lentical to the sequences of the primers used in the PCR
reaction. These primers flank the seq~ e to be ~mplifi~i which conl~lises the target sequence. Primers are typically less than about 35 nucleotides in length,5 but may be smaller or larger as n~s.~ry to generate the nucleic acid. Althoughnot required, the sequences of the primers are generally known for the primers to specifically hybridize to a relatively unique portion of nucleic acid and generate an j(l~ntifi~hle nucleic acids on PCR amplification. PCR products can be of most any length and can be distinguished from non-specific and undesired 10 amplification products by size.
In PCR and any polymerase ~mplifi(~tion procedure, extensions may be added to the S'-termini of a primer to permit post-amplification manipulations of the product without si~nific~ntly effecting the amplification reaction. These S' extensions may be restriction enzyme recognition-sites, 15 structural sequences or other seqllen~ec desirable for the process. Briefly, template DNA is first denatured by heating in the presence of a large molar excess of each of the two oligonucleotides and the four dNTPs. The reaction mixture is cooled to a temperature that allows the oligonucleotide primer to anneal to target sequences, after which the ~nn~lP~l primers are extended with 20 DNA polymerase. The cycle of dellaluldLion, ~nnt-~ling, and DNA synthesis, the principal of PCR amplification, is repeated many times to generate large qu~nfiti~s of product which can be easily identified. This tt~ e~ture cycling is made possible by the use of a DNA polymerase that does is not destroyed at the higher temperatures required for dellaLul~lion. Nucleic acid polymerases 25 which can be use~ for amplification include both DNA and RNA polymerases Many useful thermostable polymerases for PCR amplification are commercially available such as Taq DNA polymerase (Stratagene; La Jolla, CA) and AmpliTaq DNA polymerase (Perkin-Elmer Cetus; Norwalk, CT).
W O96/36731 PCT~US96/06527 The major product of this exponential reaction is a segment of double stranded nucleic acid, easily converted to single strands by, for example, rhPmir~l, pH or heat del~Lu~ilLion, whose termini are defined by the 5' termini of the oligonucleotide primers and whose length is defined by the fli~t~nre 5 between the primers. Under normal reaction conditions, the amount of polymerase becomes limiting after 25 to 30 cycles or about one million fold amplification. Further amplifir?tion is achieved by diluting the sample 1000 fold and using it as the template for further rounds of amplification in anotherPCR. By this method, amplification levels of 109 to 101~ can be achieved during 10 the course of 60 sequential cycles. This allows the detection, by hybridization with r~-iio~r~ive probes, of a single copy of the target sequence in the presence co..~;....i.-~l;..g DNA. Without the use of seq~lPnti~l PCR, the practical ~letPcti- n limit of PCR can be as low as 10 copies of DNA per sample.
Although PCR is a reliable method for amplifir~tion of target 15 sequences, a number of other techniques can be used such as isothermic amplification, ligase chain reaction (LCR), self s--ct~inPA seq~nre replication (3SR), polymerase chain reaction linked ligase chain reaction (pLCR), gaped ligase chain reaction (gLCR), ligase chain detection (LCD). The principle of ligase chain reaction is based in part on the ligation of two adjacent synthetic20 oligonucleotide primers which uniquely hybridize to one strand of the target DNA or RNA. If the target is present, the two oligonucleotides can be covalently linked by ligase. A second pair of primers, almost entirely complementary to the first pair of primers is also provided in a ligase chain reaction. In a ligase chain reaction, the template and the four primers are placed 25 into a thermocycler with thermostable ligase. As the temperature is raised and lowered, oligonucleotides are renatured adjacent to each other on the template and ligated. The ligated product of one reaction serves as the template for a subsequent round of ligation. The presence of target is manifested as a DNA
CA 02221467 1997-ll-18 W O96/36731 ~ PCTIUS96/06527 fragment with a length equal to the sum of the two adjacent oligonucleotides.
Additional PCR variations include in situ PCR and imm-mo-pCR amplification which utilizes nucleic acid fr~gmtontc coupled to pathogen-specific antibodies to increase detection sensitivity. Alternatively, nucleic acids can be analyzed after 5 purification using, for example, DNA or RNA polymerases, PCR or another amplification technique. PCR analysis of RNA, or RT-PCR, involves reverse transcription of RNA, such as mRNA sequences, into cDNA copies. These target cDNA sequer~r~s are hybridi_ed to primers which ampli~y the nucleic acid using PCR amplification.
Although high level amplification may be possible, it may not always be n~PC~, y or even desired when, for example, tLc sequence amplified is likely to mutate or otherwise be altered during the amplification process. Insuch cases, PCR can be limited to just a few rounds of amplification or avoided altogether and sequence replicated using more conventional nucleic acid 15 polymerases.
The sequence of the nucleic acid including the target sequence will be .1~ P~ by the sequence of the nucleic acid obtained from the sample.
However, synthetic seq~lenr~s may be added or the entire nucleic acid may be synth~tir~lly synth~si7~1 As such, nucleic acids may comprise any conlbi,~lion 20 of purines or pyrimi~lines, mo~ifir~tions or derivatives of purines or pyrimidines, or other ch~mir~l moieties which can be hybridi_ed specifically or non-specifically to a nucleic acid sequence. For example, neutral bases, those bases which non-specifically hybridi_e to most any other base, such as inosine or modifications or derivatives of inosine, can be incorporated. In addition, 25 incorporation of residues such as thiolated bases, boronated bases, polyamides and peptide nucleic acids can produce sequences which are resistant to Cll 'ylllaLiC
degradation.
Sequences of the nucleic acid, including the target scquence, may encode protein or be entirely non-coding sequences such as structural sequences or seqllrnr~c which regulate expression. Structural sequences include ribosomal RNA and telomeres. Controlling sequences include promoter sequences, S enh~nr~rs, 5'- and 3'-untr~n~l~ted sequences and sequences tl~at function outside of expression such as ribozymes. Identific~tion of variations within such sequences can be important in determining Llc:allllcnt regimr-nts, such as in ideneifying repeat numbers, in d~lellnil~illg molecular structure and in generating relationships. For example, target sequences within the nucleic acid may be 10 sequences which are specific to a particular species or strain of organism such as a ba~;Leliul,l, virus, parasite or fungus, or the sequence of a tr~ncl~t~d oruntr~n~l~ted portion of a eukaryotic or prokaryotic gene. Identifir~tion of suchsequences can be used to detect and often identify the org~ni~m Alternatively, the target sequence may c~ e a homologous seq~l~nr~ such as inosine, uracil 15 (U) or deoxyuracil (dU), when only the length of the target sequence is to be determin Nucleic acids are hybridized to an array of probes by any ~lumbel of techniques known to those of ordinary skill in the art. For example, hybridizations may be performed in a buffered salt solutions such as SSC (3M
20 NaCl, 0.3 M Na Citrate, pH 7.0), or SSPE (3M NaCl, 0.2 M Na Phosphate, 0.0~ M EDTA, pH 7.4). Other solutions can be utilized where melting temperature of the double helix is independent of base composition and dependent only on length. Solutions which have this property include solvents cont~ining quaternary alkylammonium salts such as solutions of tetramethyl-25 ammonium chloride or tetraethylammonium chloride. In quaternaryalkylammonium solutions the bonding strength of ~T base pairs and GC base pairs are approximately the same.
Probes of the array each comprise regions which are compl~ to one or more portions of the nucleic acid. Preferably, probes comprise 5'-region and 3'-regions which are complementary to portions of the nucleic acid and an internal variable region. The variable region can vary in 5 sequence and/or length and, preferably, one of the variable region sequences of the array is complt-m~ont~ry to or will otherwise completely hybridize to the target seq lenr~. Variations in probe sequence will prevent certain of the probes from fully hybridizing to the nucleic acid cont~ining the target sequence. Theseheteroduplexed probes, conr~ining an unhybridized portion in either the probe 10 or the nucleic acid, are susceptible to digestion using a single-strand specific nuclease.
Probes and nucleic acids may be i(lentir~lly or dirr~ Lially labeled with ~letr~t~hle labels. Detectable labels include radio-isotopes such as '25I, 35S, 32p or 3H, stable-isotope or ch~mir~l moieties such as a fluorescent,15 l~ i..rsc~ or chemilllmint~scrnt compounds. Additional labels which may be used include chromogenic chemicals, metals, coupling agents such as biotin/streptavidin or avidin, mass modifying moieties, m~gn~tic agents or chrmir~ et~o~t~hle by nuclear m~nPtir rrson~nre or electron spin resonance.
Labels may be incorporated enzyrn~tir~lly, for example, during gt;ll~.alion of the 20 nucleic acid or by rh-omic~l m~lifir-~tion of the final structure. Specifically useful labeling compounds are those which do not ..~ relc with the polymerase reaction such as rhodamine, fluorescein, dansyl chloride, coumarin, digoxin, fluoresc~minP and derivatives and modifications of these compounds.
Probes or target nucleic acids may also be fixed to a solid support 25 or free in solution. When free in solution, hybridization may be in an ordered fashion such as in well separated wells of a microtiter dish or multi-well chip,or together in a single well or small number of wells. In this fashion, batch analysis of hybrids can be performed sequentially to minimi7~ the number of ~ probes needed to identify an unknown target sequence. ~Iternatively, probes can be hybridized to nucleic acids in an ordered fashion such that individual hybridization events can be accurately scored. Useful solid supports include plastics, glasses, ceramics, metals, resins, gels, membranes, chips such as 5 hybridization chips, and combinations of these materials and structures.
This hybridized array, either fixed or free in solution, is digested with a single-strand specific nuclease to cleave single stranded regions such asheteroduplexes and terminal extensions. Nucleases suitable for digestion of hybridized probes include those nuclease which plcfe~cllLially cleave single-10 stranded nucleic acids. P~efe--cd nucleases include the endonucleases such asSl mlrle~e, mung-bean nllc le~ce, ribom-cle~e A and ribon--rle~e T1. Nucleic acids or probes which generate l~ single strands can be digested with exon--rle~es such as the T4 and T7 phage nucleases. When desired, tre?,tm~nt with excess mlrle~e can be directed to produce double-stranded cleavage by 15 eYt~n~ling the nick to a gap and thereby creating a single-stranded region on the opposite strand. Such double-stranded cuts can be useful in procedures where probes are fr~gmrnt~
Nicked hybrids can be labeled using tennin~l deo~L al~rc-~se or another suitable nucleic acid modifying enzyme, and precursor dNTPs or ddNTP
20 d~tert~bly labeled with a radio isotope, stable-isotope or chemical moiety such as a~ fluorescent, l lminrscent or ch~-mihlmin~scent moiety. Additional labels which may be incorporated include chromogenic ch~mir~l~, metals, coupling agents such as biotin/streptavidin or avidin, mass modifying moieties, m~gn.otiragents or ch.omic~ etect~ble by nuclear m~gntotic resonance or electron spin 25 resonance.
Digested hybridized probes are then contacted with a nucleic acid polymerase to extend nicked strands and thereby displace one strand of the heteroduplex. Polymerases which can be used for elongation include any polymerase which can elongate a template after a nick. Most DNA polymerase of most org~nicmc are suitable for the practice of this invention. Examples of suitable polymerase include human DNA polymerase I, II, and m, E. coli DNA
polymerase I, II, and m, T7, T3, and SP6 polymerase, thermostable DNA
5 polymerase, sequenase, and amplitaq polymerase.
An~her embodiment of this invention is directed to a method to measure the length of a target sequence. Probes constructed for length mea~u~cll~ents preferably comprise neutral bases such as inosine residues flanked by two constant region sequences. An advantage of neutral bases in that a 10 knowledge of the target sequence is not required. Neutral base forms stable base pairs with all four conventional bases and the strength of the paring is approximately equal in each case. With the use of a neutral base, the assay willbe sensitive only to the length, but not the sequence of the target.
Another embodirnent of the invention is directed to a method for 15 detecting the number of repeat sequences in a target nucleic acid. A target sequence may be from a natural source or a synthetic source. Natural sources of target sequence may include DNA, and RNA from an org~ni~m The nucleic acid may be from seq l~onrPs which encodes a protein, such as exons and mRNA.
The nucleic acid may also be from structural and from non-coding sequences 20 such as ribosomal RNA, and telomeres. Genes which comprise repeated sequences, such as human TFIID and human DNA polyrnerase II largest subunit, have internal trinucleotide repeats which encodes for strings of homopeptides whose length varies between individuals. Non coding repeat sequences include the repeating DNA and telomeric sequences. Synthetic sources of nucleic acids 25 may be from a laboratory reaction, a nucleic acid synthesis machine. Additional sources of nucleic acids may be from nucleic acids added to industrial and consumer goods.
-W O96/36731 PCTrUS96/06527 To determine the number of repeats in a target sequence, the target sequence is hybridized to a plurality of probes, each cont~ining none, one or more than one repeat. Where the number of repeats in the target do not correspond to the number of repeats in the probe, one or more single stranded S loop can be present on the target-probe hybrid. Single stranded loops are onlyabsent in the hybrid with a perfect match. Perfect m~tch~s co..~ hybrids of nucleic acid target to probes with the same number of repeats. Single strand nuclease treatrn~qnt after hybridization will digest all the single stranded loops leaving nicked hybrids and un-nicked hybrids. Polymerase tre~tmrnt after 10 digestion elongates and displaces strands of all nicked hybrids. Hybrids with a perfect match and without nicks will be the only hybrids not affected by polymerase. By m~ o. ill~, the polymerase reaction, the hybrid with the perfect match can be identified and the number of repeats in the target can be detel...i..~ cl.
The polymerase reaction can be monitored by a llu~ el of mrthod~. The polymerase elongation reaction may be ~,r~ ed in the presence of nucleotide triph~ sph~ttos with a detect~hle moiety. On ~letPct~hle moiety is a radio-label such as 32p or 35S on the a-phosph~tr. All the hybrids with an incorrect number of repeated se~enr-e will be labelled while the hybrid with 20 equal number of repeats will remain unlabeled. Thus, the assay allows for theprecise identifir~tion of the numher of bases or the number of repeat seq~rnres in a target sequence. As such, these methods are faster and more sensitive than methods currently available.
Another embodimem of the invention is directed to a method for 25 screening a patient suspected of having a genetic disorder. A sample of tissue is obtained such as a sample of tissue or bodily fluid, and nucleic acid PCR
amplified, purified or cloned. The target nucleic acid sequence is hybridized toan array of probes, nuclease and polymerase treated and the presence or absence CA 02221467 1997-ll-18 W O96/36731 PCTrUS96/06527 of the genetic defect ~l~tect~d. Disorders which can be ~lettocte(l include, forexample, lllyotonic dystrophy, ~llntington~ s disease, Kennedy disease and Fragile X ~ylldlulllc. Patients may be any m~nnm~l such as a human. Patient s~mplç~ may be collected and pooled to reduce the number of tests which need to be performed to identify a positive carrier, or sequentially analyzed againsta variety of different probe arrays to further limit the number of tests and probes needed.
Another embodiment of the invention is directed to arrays of probes wherein each probe comprises a constant 5'-region, a constant 3'-region 10 and a variable internal region wherein the variable region comprises one or more repeat sequences. The repeat sequence comprises heterologous or homologous sequences which are variable in length or base sequence. Sequences contain purine or pyrimi~lin~ bases or neutral bases such as inosine. Either the nucleicacids or the probes of the array may be labeled with a detect~hle label or-fixed15 to a solid support. Arrays may be spatially ordered by structure or sequence with the seq~l~n~s of the probes known or ~ hle. Probes may be single-stranded or partly single-str~nrl~ and partly double-stranded. Probes may also be labeled with dett~ct~ble labels. Arrays may comprise between about 10 to about 10,000 dirr~lelll probes, preferable between about 50 to 5000 dirrelclll 20 probes, or more or less as required.
The following e~.illlents are offered to illustrate embo lim~n of the invention, and should not be viewed as limhing the scope of the invention.
Fxamples Example 1 Oli~onucleotide Syn~hesis. Purification. and Characl~ tion.
Synthetic oligonucleotides comprising the following sequence were synthesized using an oligonucleotide synthesizer (Operon Technologies, Inc.). The sequences of the oligonucleotides are as follows:
W O96/36731 PCTrUS96/06~27 Tl(78 mP~
5'-CCAGATCTGA TGCGTCGGAT CATCCAGCAG CAGCAGCAGC
AGCAGCAGTC ACGCTAACCG AATCCCTGGT CAGATCTT-3' (SEQID NO 1) T2(78 mer) S'-AAGATCTGAC CAGGGATTCG GTTAGCGTGA CTGCTGCTGC
TGCTGCTGCT GCTGGATGAT CCGACGCATC AGATCTGG-3' (SEQID NO 2) CTG6(72 rn~r) S'-AAGATCTGAC CAGGGATTCG GTTAGCGTGA CTGCTGCTGC
TGCTGCTGGA TGATCCGACG CATCAGATCT GG-3' (SEQID NO3) Oligonucleotides T1 and T2 were purified by polyacrylamide gel electrophoresis, while CTG6 was purified by using high pelrcllllaLce liquid 15 cl~,o~a~ography. The concentration of each stock solution was ~leterrnin~l by absorption at 260 nm.
Tl,T2 and CTG6 contain 8 GAC repeats, 8 CTG repeats, 6 CTG
repeats, respectively. The GAC repeats are located 30 bases from the 5' end and 24 from the 3' end. The CTG repeats are located 24 from the 5' end and 20 30 from the 3' end.
Example 2 Deternlin~tion of S1 Nuclease Specificity ~n-l F.fficienry.
S1 mlcle~e specificity and effirien~y was monitored using 5' radio-labeled oligonucleotides. Briefly, 3.5 ,uM of oligonucleotide was placed in kinase buffer (70 rnM Tris-HCl, pH 7.6,10 rnM MgCI2, 5 mM dithiothreitol) 25 cont~inin~ 6.4 pM 3~P-ATP (specific activity of60 Ci/mrnole). End labeling was initi~t~l by the addition of 0.35 unit/pmole oligo T4 polynucleotide kirlase(New Fn~l~n~l Biolabs; Beverly, MA). Labeling continnecl for 45 mimlt~s at 37~C. Labeled oligonucleotides were separated from unincorporated 32P-ATP
with a CHROMA-SPINTM+TE 10 columns (Clonetech).
CA 02221467 1997-ll-lX
W O96/36731 PCTrUS96/06527 Heteroduplexes were g~ t~d by ~nnr~ling 1 ,uM of 32P-labeled oligonucleotide T1 to an equal molar amount of T2 or CT66 in a 50 pL volume of 100 mM Tris-HCI, pH 8.0 (Figure 1). Oligonucleotides were heated to 96~C
for four ...i....~ and gradually cooled to 30~C over two hours to ensure specific S ~nn-o~ling.
The specificity of S1 as a function of enzyme concentration was tested using T1-T2 and Tl-CTG6 heteroduplexes labeled as Hl and H2, respectively, in Figure 1. Briefly, 0.1-1.0 unit/picomole of S1 nuclease (Promega; Madison, WI) was added to the heteroduplexes in a solution of 200 10 mM NaCI, 50 mM sodium acetate, pH 4.5, 1 mM ZnSO4, 0.5% glycerol.
Nuclease digestion was ~lr~ ed at temperatures of abou. 0~C, about 24~C and about 37~C. The L~lll~lalul~S of the solutions were equilibrated to the reactiontelllpelàture before the addition of enzyme. After a reaction period of 60 minlltes, further digestion was stopped by the addition of EDTA to a final 15 concentration of 12 mM. .Sch~m~tirs of the expected reaction products are shown in Figure 1 C, and D. Each reaction product was analyzed by native 12% polyacrylamide gel electrophoresis. R~cllltin~ gels were autoradiographed and are depicted in Figures 2A and 2B. Figure 2A depicts an autoradiograph of the reaction product of the perfect match heteroduplex T1-T2. T ~ne 1 is a minus20 S1 control. Lanes 2-5 contain increasing collcellLIalions of Sl (0.2, 0.5, 0.8, 1.0 units per picomole oligo) all incubated at 0~C. Lanes 6-9 contain identical concentrations, but were inr~h~ted at room telll~elalule, and lanes 10-13 were incubated at 37~C. Although at higher temperatures S1 cut the end label off of the duplex, no other cutting was seen. Lane 0 contains size standard 25 (~X174/HinfI digest).
Figure 2B is an autoradiograph of the reaction product of the mi~m~tched heteroduplex T1-CTG6. Lanes 1-4 contained increasing concentrations of Sl (as above), all inr~lb~ted at 0~C. Lanes 5-8 follow the W O96/36731 PCTrUS96/06527 same pattern of S1 concentration, but were in~lb~tP~ at room temp~rature, while lanes 9-12 were inr~lbatP~ at 37~C. Both lanes 13 and 14 contain T1-CTG6 complex without any Sl mlclP~e. The top band in each lane (band A) m~tchPs with the T1-CTG6 control and is just the uncut loop structure. The second band 5 (band B) is the nicked loop, while band C appears to be a nicked loop that hasbeen partially digested. Lane D is very faint, but may contain completely digested loop, leaving a nicked duplex DNA. T ~ne 15 contains a size standard.
At 0~C, greater than about 60% of the 6 base loops gel~eratcd by the mi~m~tchPA repeats in the Tl-CTG6 hybrid complex were cut by S1 nuclease 10 at a concentration of 0.6 units per picomole (Figure 2). The presence of multiple bands was most likely due to S1 nn~le~e cleaving the loop structure and thereby degrading several unpaired nucleotides. It also appears that S1 nuclease cut several u~ail~d nucleotides rather than just one, since distinct bands appeared at separations of more than one base pair. In contras~, no 15 cleavage was seen with the pc,re~;lly m~t~hP~l Tl-T2 hybrid complex.
At higher (~ l cs7 less of the label ap~cd in each lane of both the m~tchP~l and ...~ hPCl samples. This was most likely due to Sl nuclease cleaving the b-call~illg ends of duplex DNA as single-stranded structures were formed. This problem was not seen in samples inrnb~tPd at 0~C
20 because the extent to which the DNA ends could breath was reduced. These experiments ~lemo~ aled that S1 nllcle~ce cleaved the hybrid cont~ining a micm~tch at the location of the mi~m~trh Example 3 T ~helir~ ~n-l Strand Displacement.
An enh~n~e~l method to discriminate between the m~tfhP-l and 25 mi~m~tchPA oligonucleotides was e~minPA . Labeling and strand displ~- emPnt reactions were tested with templates con~icting of unlabeled T1-T2 and T1-CTG6 heteroduplexes. Digestion of these duplexes was performed with 0.6 units of S1 nuclease per picomole of oligonucleotide at 0~C. Reactions were W O96/36731 PCTrUS96/06527 ter~i~L1ated and the products purified with a spin column (CHROMA-SPI~M+TE 10). S1 nllcle~ce was inactivated after column purification of the oligonucleotide because of the removal of ZnSO4.
The ex~. ~l~..L~l scheme and the expected results are represented 5 in Figure 3. The expected digestion products of the mi.cm~tch~l heteroduplex is represented as Al while the expected digestion product of the perfect match heteroduplex is represented as A2. The expected reaction product after polymerase treat~lnent is shown as B1 and B2, respectively.
Labeling of the Sl digested heteroduplexes were performed for 10 15 ~ s at room t~lll~ldtU~ with the Klenow fragment of DNA polymerase I. Briefly, 0.08 units per picomole of enzyme was added in a reaction buffer of 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 0.001% gelatine, 30 pM of each dNTPs, and 32P-labeled dCTP (specific activity of 1.74 Ci/mmole) in a volume of 50 pl. The reaction was stopped by addition of sodium dc~decyl 15 sulfate (SDS) to a final concentration of 0.5%.
The product of the labeling reaction was analyzed by ac.-ylamide gel electrophoresis and autoradiography. A copy of the autoradiograph is shown in Figure 4. Lane 0 is a molecular weight m~rker. Lane 1 and Lane 2 represents S1 digested and polymerase treated micm~t(~.h.--l heteroduplex 20 elongated in the presence (lane 1) and absence (lane 2) of radioactive nucleotide triphosphates. Lane 3 and lane 4 represents S1 digested a~d polymerase treated perfect match heteroduplex elongated in the presence (lane 3) and absence (lane 4) of radioactive nucleotide triphosphates.
Incorporation of 32P-labeled of dCMP in the S l-cleaved, 25 mi.cm~s~hed hybrid (Tl-CTG6) by Klenow fr~gm~nt yielded a strong signal at the position expected if the S1 cleavage occurred at the site of the micm~s~.h (Figure 4). Omy a very weak signal could be ~let~-ct~ for t'ne perfectly m~t~h~lhybrid (Tl-T2), and this signal was not localized into any distinct bands. Some CA 02221467 1997-ll-18 W O96/36731 PCTrUS96/06527 non-specific labeling of the perfectly matched hybrid, as ~vell ac the T1-CTG6 complex may have arisen from the t~?n~l.on(~y for Sl nuclease to introduce nicksinto double-stranded DNA. However, the loop-cutting activity of Sl nuclease is much stronger than its ability to introduce nicks into perfectly m~trhtocl 5 double-stranded DNA, which is demonstrated in these experiments.
Example 4 Detection of a Repeated Genomic Sequence.
A single-stranded nucleic acid co~ fl~ g an internal target repeat sequence is generated from genomic DNA for analysis. A sch~ tic of the strategy is shown in Figure 5. Briefly, one 5'-biotinylated oligonucleotide 10 primer and one non-biotinylated primer is produced using an oligonucleotide ~y~ r. The ~ el:i flank a region of genomic DNA cont~ining a variable number of repeated nucleotides. A polymerase chain reaction is performed using the two primers and genomic DNA as template (Figure SA). Double stranded reaction product is purified from unincorporated nucleotide 15 triphosphates by a size eY~ cion column. The purified PCR product is de~ ued in 8M urea and the biotinylated strand removed. The non-biotinylated strand is labeled at the 3' end with a fluolesc~ill and used as the target nucleic acid.
A plurality of probes, each cont~ining 5' and a 3' sequence 20 comple.~ to the target nucleic acid and from 10 to 109 internal repeats are synthPci7~oA on an oligonucleotide synth~--ci7~r. Probes of 80 bases or shorter are synth~ci7~l and used directly. Probes greater than 80 bases in size are synthesized as fr~mentc and ligated together. After generation, probes are labeled at the 3' ~ lc with rho~minP. All the probes are synth~ci7e~ with 25 a 5' biotin and these biotinylated probes are ~tt~ch~d to the bottom of a plate coated with immobilized streptavidin. Probes are attached along a 10xlO array and ordered according to size (Figure SB).
WO96/36731 PCT/US96/06~27 Target nucleic acid is hybridized to the probe array (Figure SC) and digested with S1 nuclease (Figure SD). DNA polymerase is added to the array and elongation and strand displ~r~m~-nt is allowed to occur (Figure SE) until completion (Figure SF). When the probe contains more internal repeats S than the target, the rh~l~mine label will be lost in the strand displ~em~nt and the r~-slllt~nt proa-lct will be red. Similarly, when the target contains more internal repeats than the probe, the fluorescein label will be lost and the product will be green. When the probe and the target both contain the same number of repeats, both rhodamine and fluolcsceill will remain and the reslllt~nr color will 10 be yellow.
After strand displacement the array is inspected visually. The result is displayed in Figure 6. All the probes are yellow before strand displ~ment (Figure 6A). After S1 cutting and strand displ~f~m.ont, the probes with fewer repeats than the target is red and the probe with more repeats is lS green. The probe with the sarne number of repeat is yellow. The results of expe~ c~ clrolllled with the same probe array but with target DNA
comprising 88, 55, and 17 repeats are shown in Figure 6B. This experiment demonstrates how a colormetric assay may be performed to delcl~ e the nurnber of repeats in a target sequence.
20 Example S Detection of Repeated Seque~o from Myotonic Dystrophy Patient.
To (~ the extent of expansion of trinucleotide repeat in a myotonic patient, a S ml sample of blood is drawn from the patient for ~n~lysis.Whole cell DNA is isolated from the blood and a DNA, comprising a region of 25 trinucleotide repeats, implicated as a cause for myotonic dystrophy disorder, is amplified and isolated by polymerase chain reaction. Polymerase chain reaction products are denatured and one of the DNA strands used as the nucleic acid cont~ining the target sequence to be detected.
CA 0222l467 l997-ll-l8 W O96/36731 PCTrUS96/06527 ~ An oligonucleotide synthesizer is used to generate a set of oligonucleotide probes. Each probe in the set has a 20 base-pair 5' sequence anda 20 base-pair 3' sequence compleTnPnt~ry to the sequence fl~nking the trinucleotide repeat region. In addition, each probe in the set has an internal 5 trinllrlPotide repeat between the 5' and 3' sequence. A series of 20 probes are synthesized cont~ining from 1 to 20 trinucleotide repeats.
Three picomoles of each probe, a total of 60 picomoles, is hybridized to 200 pmoles of the amplified target nucleic acid. Briefly, the probes and the targets are heated in 100 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 to 96~C for four minllt~Ps and cooled gradually to 30~C over two hours to ensure specific ~nnP~Iing to form heteroduplex with mi.~m~tc~ s and perfect m~t llPs.
Heteroduplexes are treated with 0.3 unit per picomole of Sl ml~le~e at 0~C for S mimltes. The reaction is stopped by chromatography of the reaction ~ we through a spin column.
Polymerase tre~tmPnt of the Sl digested heteroduplexes is performed for 15 ..ii....~es at room l~u~ d~ul~ with the Klenow fragment of DNA polymerase I. Briefly, 0.08 units of enzyme is added per picomole DNA
in a reaction buffer of 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 0.001% gelatine, 30 pM of each dNTPs. The reaction is stopped by ~ lifi~n of sodium dodecyl sulfate (SDS) to a final collcel.l.dlion of 0.5%.
The product of this reaction is analyzed on a delldlul ing sequencing gel with the set of DNA probes as a molecular weight marker. After electrophoresis, the gel is treated with water for 30 minutes to remove the ureaand stained with SBYR or FBIR. Bands are ~ tected upon exposure to ultraviolet light. The largest product observed is a 61 base band corresponding to 7 trinucleotide repeats.
Other embo lim~ontc and uses of the invention will be apl)alelll to those skilled in the art from consideration of the specification and practice of t;le W O96/36731 PCTrUS96/06527 invention disclosed herein. All U.S. Patents cited herein are specifically incorporated by lt;r~lellce. The specification and examples should be consideredexemplary only with the true scope and spirit of the invention in-1ir~te~1 by the following claims.
s
Claims
We claim:
1. A method for detecting a target sequence within a nucleic acid comprising the steps of:
a) hybridizing the nucleic acid to an array of probes wherein each probe comprises a 5'-region complementary to said nucleic acid, a 3'-region complementary to said nucleic acid and an internal variable region;
b) digesting the hybridized array with a single-strand specific nuclease;
c) treating said array with a nucleic acid polymerase; and d) detecting the target sequence.
2. The method of claim 1 wherein the target sequence comprises a plurality of repeat sequences.
3. The method of claim 2 wherein the plurality comprises between about 2 to about 2000 repeat sequences.
4. The method of claim 2 wherein the repeat sequences are each between about 2 to about 25 nucleotides in length.
5. The method of claim 1 wherein the nucleic acid is DNA, RNA, PNA or modifications or derivatives thereof.
6. The method of claim 1 wherein the nucleic acid is labeled at a 5'-terminus or a 3'-terminus with a detectable label.
7. The method of claim 6 wherein the detectable label is selected from the group consisting of radio isotopes, stable isotopes, luminescent and electroluminescent chemicals, fluorescent chemical, chromogenic chemicals, metals, coupling agents and magnetic agents.
8. The method of claim 1 wherein the nucleic acid is derived from a biological sample.
9. The method of claim 8 wherein the biological sample is a sample of biomass, biodily tissue, fluid or combination thereof.
10. The method of claim 1 wherein the nucleic acid is a polymerase chain reaction product.
11. The method of claim 1 wherein the 5'-region and the 3'-region are each between about 15 to about 100 nucleotides in length.
12. The method of claim 1 wherein the variable region comprises a plurality of repeat sequences.
13. The method of claim 12 wherein the plurality comprises between about 2 to about 2000 repeat sequences.
14. The method of claim 13 wherein the repeat sequences are each between about 2 to about 25 nucleotides in length.
15. The method of claim 1 wherein the variable region is variable in sequence or length.
16. The method of claim 15 wherein the length is between about 10 to about 2000 nucleotides.
17. The method of claim 1 wherein the variable region comprises a sequence of neutral bases.
18. The method of claim 17 wherein the neutral bases are inosine bases.
19. The method of claim 1 wherein the nucleic acid and the probes are labeled with different chromogenic chemicals.
20 . The method of claim 1 wherein the array is fixed to a solid support.
21. The method of claim 20 wherein the solid support is selected from the group consisting of plastics, glasses, ceramics, metals, resins, gels, membranes, chips and combinations thereof.
22. The method of claim 1 wherein the single-strand specific nuclease is S1 nuclease, mung bean nuclease, ribonuclease A or ribonuclease T1.
23. The method of claim 1 wherein the nucleic acid polymerase is a DNA
polymerase, a reverse transcriptase, an RNA polymerase or a thermostable polymerase.
24. The method of claim 1 wherein the target sequence detected is indicative of a disorder.
25. The method of claim 1 wherein the disorder is myotonic dystrophy, Huntington's disease, Kennedy disease or Fragile X syndrome.
26. The method of claim 1 wherein the nucleic acid comprises a plurality of different nucleic acids.
27 The method of claim 26 wherein the plurality is fixed to a solid support.
28. The method of claim 27 wherein different probes of the array are hybridized to the fixed nucleic acids sequentially.
29. A method for determining a length of a target sequence within a nucleic acid comprising the steps of:
a) hybridizing the nucleic acid to an array of probes wherein each probe comprises a 5'-region complementary to said nucleic acid.
a 3'-region complementary to said nucleic acid, and an internal variable region;
b) digesting the hybridized array with a single-strand specific nuclease;
c) treating said array with a nucleic acid polymerase; and d) determining the length of the target sequence.
30. The method of claim 29 wherein the nucleic acid is a PCR product.
31. The method of claim 29 wherein internal variable region comprises a homologous sequence of bases.
32. The method of claim 31 wherein the homologous bases are inosine residues or modifications or derivatives of inosine residues.
33. A method for determining a number of repeat sequences within a nucleic acid comprising the steps of:
a) hybridizing the nucleic acid to an array of probes wherein each probe comprises a 5'-region complementary to said nucleic acid, a 3'-region complementary to said nucleic acid and an internal region containing one or more repeat sequences;
b) digesting the hybridized array with a single-strand specific nuclease;
c) treating said array with a nucleic acid polymerase; and d) determining of number of repeat sequences within the nucleic acid.
34. The method of claim 33 wherein the nucleic acid is derived from a mammal, an insect or a microorganism.
35. The method of claim 33 wherein the array comprises greater that R
different probes and R is the number of repeat sequences in the target sequence.36. The method of claim 33 wherein the array comprises a fraction of R
probes and R is the number of repeats in the target sequence.
37. The method of claim 36 wherein the steps a, b, and c are repeated using a difference fraction of the array, 38. The method of claim 33 wherein the internal region is between about 10 to about 2000 nucleotides in length.
39. The method of claim 33 wherein the repeat sequences are each between about 2 to about 10 nucleotides in length, 40. The method of claim 33 wherein the repeat sequences are contiguous.
41. The method of claim 33 wherein the neutral bases are inosine residues or modification or derivatives of inosine residues.
42. The method of claim 33 further comprising the step of pooling a collecting of different nucleic acids and hybridizing the collection to the array.
43 A method for screening a patient suspected of having a genetic disorder comprising the steps of:
a) obtaining a tissue sample from said patient;
b) amplifying a target sequence of said sample;
c) hybridizing said target sequence to an array of probes wherein each probe comprises a 5'-region complementary to said nucleic acid, a 3'-region complementary to said nucleic acid and a variable internal region;
d) digesting the hybridized array with a single-strand specific nuclease:
e) treating said array with a nucleic acid polymerase; and f) detecting the presence of absence of the genetic disorder.
44. The method of claim 43 wherein the patient is a mammal.
45. The method of claim 44 wherein the mammal is a human.
46. The method of claim 43 wherein the genetic disorder is myotonic dystrophy,Huntington's disease. Kennedy disease or Fragile X syndrome.
47. The method of claim 43 wherein the nucleic acid is amplified by polymerse chain reaction.
48. The method of claim 43 further comprising the step of pooling a collection of nucleic acids from different patients, hybridizing the collection to the array and determining the present or absence of the genetic disorder in any of the patients.
49. An array of probes wherein each probe comprises a constant 5'-region, a constant 3'-region and a variable internal region wherein said variable regioncomprises one or more repeat sequences.
50. The array of claim 49 wherein the repeat sequence comprises a sequence of inosine residues.
51. The array of claim 49 which comprises between about 50 to about 5000 different probes.
52. The array of claim 49 which comprises greater than 5000 different probes.
53. The array of claim 49 which comprises DNA, RNA, PNA or modifications or derivatives thereof.
54. The array of claim 49 wherein the probes are labeled with a detectable label.
55. The array of claim 54 wherein the detectable label is a chromatic chemical.
56. The array of claim 49 which is fixed to a solid support.
1. A method for detecting a target sequence within a nucleic acid comprising the steps of:
a) hybridizing the nucleic acid to an array of probes wherein each probe comprises a 5'-region complementary to said nucleic acid, a 3'-region complementary to said nucleic acid and an internal variable region;
b) digesting the hybridized array with a single-strand specific nuclease;
c) treating said array with a nucleic acid polymerase; and d) detecting the target sequence.
2. The method of claim 1 wherein the target sequence comprises a plurality of repeat sequences.
3. The method of claim 2 wherein the plurality comprises between about 2 to about 2000 repeat sequences.
4. The method of claim 2 wherein the repeat sequences are each between about 2 to about 25 nucleotides in length.
5. The method of claim 1 wherein the nucleic acid is DNA, RNA, PNA or modifications or derivatives thereof.
6. The method of claim 1 wherein the nucleic acid is labeled at a 5'-terminus or a 3'-terminus with a detectable label.
7. The method of claim 6 wherein the detectable label is selected from the group consisting of radio isotopes, stable isotopes, luminescent and electroluminescent chemicals, fluorescent chemical, chromogenic chemicals, metals, coupling agents and magnetic agents.
8. The method of claim 1 wherein the nucleic acid is derived from a biological sample.
9. The method of claim 8 wherein the biological sample is a sample of biomass, biodily tissue, fluid or combination thereof.
10. The method of claim 1 wherein the nucleic acid is a polymerase chain reaction product.
11. The method of claim 1 wherein the 5'-region and the 3'-region are each between about 15 to about 100 nucleotides in length.
12. The method of claim 1 wherein the variable region comprises a plurality of repeat sequences.
13. The method of claim 12 wherein the plurality comprises between about 2 to about 2000 repeat sequences.
14. The method of claim 13 wherein the repeat sequences are each between about 2 to about 25 nucleotides in length.
15. The method of claim 1 wherein the variable region is variable in sequence or length.
16. The method of claim 15 wherein the length is between about 10 to about 2000 nucleotides.
17. The method of claim 1 wherein the variable region comprises a sequence of neutral bases.
18. The method of claim 17 wherein the neutral bases are inosine bases.
19. The method of claim 1 wherein the nucleic acid and the probes are labeled with different chromogenic chemicals.
20 . The method of claim 1 wherein the array is fixed to a solid support.
21. The method of claim 20 wherein the solid support is selected from the group consisting of plastics, glasses, ceramics, metals, resins, gels, membranes, chips and combinations thereof.
22. The method of claim 1 wherein the single-strand specific nuclease is S1 nuclease, mung bean nuclease, ribonuclease A or ribonuclease T1.
23. The method of claim 1 wherein the nucleic acid polymerase is a DNA
polymerase, a reverse transcriptase, an RNA polymerase or a thermostable polymerase.
24. The method of claim 1 wherein the target sequence detected is indicative of a disorder.
25. The method of claim 1 wherein the disorder is myotonic dystrophy, Huntington's disease, Kennedy disease or Fragile X syndrome.
26. The method of claim 1 wherein the nucleic acid comprises a plurality of different nucleic acids.
27 The method of claim 26 wherein the plurality is fixed to a solid support.
28. The method of claim 27 wherein different probes of the array are hybridized to the fixed nucleic acids sequentially.
29. A method for determining a length of a target sequence within a nucleic acid comprising the steps of:
a) hybridizing the nucleic acid to an array of probes wherein each probe comprises a 5'-region complementary to said nucleic acid.
a 3'-region complementary to said nucleic acid, and an internal variable region;
b) digesting the hybridized array with a single-strand specific nuclease;
c) treating said array with a nucleic acid polymerase; and d) determining the length of the target sequence.
30. The method of claim 29 wherein the nucleic acid is a PCR product.
31. The method of claim 29 wherein internal variable region comprises a homologous sequence of bases.
32. The method of claim 31 wherein the homologous bases are inosine residues or modifications or derivatives of inosine residues.
33. A method for determining a number of repeat sequences within a nucleic acid comprising the steps of:
a) hybridizing the nucleic acid to an array of probes wherein each probe comprises a 5'-region complementary to said nucleic acid, a 3'-region complementary to said nucleic acid and an internal region containing one or more repeat sequences;
b) digesting the hybridized array with a single-strand specific nuclease;
c) treating said array with a nucleic acid polymerase; and d) determining of number of repeat sequences within the nucleic acid.
34. The method of claim 33 wherein the nucleic acid is derived from a mammal, an insect or a microorganism.
35. The method of claim 33 wherein the array comprises greater that R
different probes and R is the number of repeat sequences in the target sequence.36. The method of claim 33 wherein the array comprises a fraction of R
probes and R is the number of repeats in the target sequence.
37. The method of claim 36 wherein the steps a, b, and c are repeated using a difference fraction of the array, 38. The method of claim 33 wherein the internal region is between about 10 to about 2000 nucleotides in length.
39. The method of claim 33 wherein the repeat sequences are each between about 2 to about 10 nucleotides in length, 40. The method of claim 33 wherein the repeat sequences are contiguous.
41. The method of claim 33 wherein the neutral bases are inosine residues or modification or derivatives of inosine residues.
42. The method of claim 33 further comprising the step of pooling a collecting of different nucleic acids and hybridizing the collection to the array.
43 A method for screening a patient suspected of having a genetic disorder comprising the steps of:
a) obtaining a tissue sample from said patient;
b) amplifying a target sequence of said sample;
c) hybridizing said target sequence to an array of probes wherein each probe comprises a 5'-region complementary to said nucleic acid, a 3'-region complementary to said nucleic acid and a variable internal region;
d) digesting the hybridized array with a single-strand specific nuclease:
e) treating said array with a nucleic acid polymerase; and f) detecting the presence of absence of the genetic disorder.
44. The method of claim 43 wherein the patient is a mammal.
45. The method of claim 44 wherein the mammal is a human.
46. The method of claim 43 wherein the genetic disorder is myotonic dystrophy,Huntington's disease. Kennedy disease or Fragile X syndrome.
47. The method of claim 43 wherein the nucleic acid is amplified by polymerse chain reaction.
48. The method of claim 43 further comprising the step of pooling a collection of nucleic acids from different patients, hybridizing the collection to the array and determining the present or absence of the genetic disorder in any of the patients.
49. An array of probes wherein each probe comprises a constant 5'-region, a constant 3'-region and a variable internal region wherein said variable regioncomprises one or more repeat sequences.
50. The array of claim 49 wherein the repeat sequence comprises a sequence of inosine residues.
51. The array of claim 49 which comprises between about 50 to about 5000 different probes.
52. The array of claim 49 which comprises greater than 5000 different probes.
53. The array of claim 49 which comprises DNA, RNA, PNA or modifications or derivatives thereof.
54. The array of claim 49 wherein the probes are labeled with a detectable label.
55. The array of claim 54 wherein the detectable label is a chromatic chemical.
56. The array of claim 49 which is fixed to a solid support.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/446,102 | 1995-05-19 | ||
| US08/446,102 US5753439A (en) | 1995-05-19 | 1995-05-19 | Nucleic acid detection methods |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2221467A1 true CA2221467A1 (en) | 1996-11-21 |
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ID=23771327
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002221467A Abandoned CA2221467A1 (en) | 1995-05-19 | 1996-05-20 | Nucleic acid detection methods |
Country Status (7)
| Country | Link |
|---|---|
| US (1) | US5753439A (en) |
| EP (1) | EP0827551B1 (en) |
| AT (1) | ATE183244T1 (en) |
| AU (1) | AU6248696A (en) |
| CA (1) | CA2221467A1 (en) |
| DE (1) | DE69603723T2 (en) |
| WO (1) | WO1996036731A2 (en) |
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| US20130183666A1 (en) | 2012-01-18 | 2013-07-18 | Marc N. Feiglin | Partial genotyping by differential hybridization |
| WO2013141331A1 (en) * | 2012-03-22 | 2013-09-26 | 和光純薬工業株式会社 | Method for detecting dna having microsatellite region |
| EP2867373B1 (en) * | 2012-06-29 | 2018-12-19 | HTG Molecular Diagnostics, Inc. | Nuclease protection methods for detection of nucleotide variants |
| US10933417B2 (en) | 2013-03-15 | 2021-03-02 | Nanobiosym, Inc. | Systems and methods for mobile device analysis of nucleic acids and proteins |
| GB2563357A (en) * | 2016-02-10 | 2018-12-12 | Nec Corp | DNA testing chip, DNA testing method, DNA testing system, and DNA testing chip control device |
Family Cites Families (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU6834187A (en) * | 1985-12-17 | 1987-07-15 | Genetics Institute Inc. | Displacement polynucleotide method and reagent complex |
| US4946773A (en) * | 1985-12-23 | 1990-08-07 | President And Fellows Of Harvard College | Detection of base pair mismatches using RNAase A |
| GB8606719D0 (en) * | 1986-03-19 | 1986-04-23 | Lister Preventive Med | Genetic probes |
| CA1341576C (en) * | 1986-08-11 | 2008-07-08 | Thaddeus P. Dryja | Diagnosis of retinoblastoma |
| US5032502A (en) * | 1988-01-21 | 1991-07-16 | The United States Of America As Represented By The United States Of Energy | Purification of polymorphic components of complex genomes |
| US5459039A (en) * | 1989-05-12 | 1995-10-17 | Duke University | Methods for mapping genetic mutations |
| AU7762091A (en) * | 1990-03-30 | 1991-10-30 | City Of Hope | Detection of minimal residual disease in lymphoid malignancies |
| DE69132843T2 (en) * | 1990-12-06 | 2002-09-12 | Affymetrix Inc N D Ges D Staat | Identification of nucleic acids in samples |
| US5455166A (en) * | 1991-01-31 | 1995-10-03 | Becton, Dickinson And Company | Strand displacement amplification |
| US5376526A (en) * | 1992-05-06 | 1994-12-27 | The Board Of Trustees Of The Leland Stanford Junior University | Genomic mismatch scanning |
| US5695933A (en) * | 1993-05-28 | 1997-12-09 | Massachusetts Institute Of Technology | Direct detection of expanded nucleotide repeats in the human genome |
| WO1995021269A1 (en) * | 1994-02-04 | 1995-08-10 | Perlin Mark K | Method and apparatus for analyzing genetic material |
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1995
- 1995-05-19 US US08/446,102 patent/US5753439A/en not_active Expired - Fee Related
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1996
- 1996-05-20 AT AT96921212T patent/ATE183244T1/en not_active IP Right Cessation
- 1996-05-20 WO PCT/US1996/006527 patent/WO1996036731A2/en active IP Right Grant
- 1996-05-20 EP EP96921212A patent/EP0827551B1/en not_active Expired - Lifetime
- 1996-05-20 AU AU62486/96A patent/AU6248696A/en not_active Abandoned
- 1996-05-20 DE DE69603723T patent/DE69603723T2/en not_active Expired - Fee Related
- 1996-05-20 CA CA002221467A patent/CA2221467A1/en not_active Abandoned
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|---|---|
| AU6248696A (en) | 1996-11-29 |
| US5753439A (en) | 1998-05-19 |
| WO1996036731A2 (en) | 1996-11-21 |
| DE69603723T2 (en) | 2000-03-02 |
| EP0827551B1 (en) | 1999-08-11 |
| ATE183244T1 (en) | 1999-08-15 |
| DE69603723D1 (en) | 1999-09-16 |
| WO1996036731A3 (en) | 1997-02-06 |
| EP0827551A2 (en) | 1998-03-11 |
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| FZDE | Discontinued |