CA2317789A1 - Method and kit for evaluation of hiv mutations - Google Patents
Method and kit for evaluation of hiv mutations Download PDFInfo
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- CA2317789A1 CA2317789A1 CA002317789A CA2317789A CA2317789A1 CA 2317789 A1 CA2317789 A1 CA 2317789A1 CA 002317789 A CA002317789 A CA 002317789A CA 2317789 A CA2317789 A CA 2317789A CA 2317789 A1 CA2317789 A1 CA 2317789A1
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- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
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- C12Q1/6813—Hybridisation assays
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
A method for obtaining information about the allelic type of a sample of genetic material derived from an HIV-infected sample relies on the observation that using a second nested set of sequencing primers ensures the maximum potential for sequencing a particular sample. To perform the method, reagents suitable for performing the tests are suitably packaged as a kit.
Description
METHOD AND KIT FOR EVALUATION OF HIV MUTATIONS
BACKGROUND OF THE INVENTION
Genetic testing to determine the presence of or a susceptibility to a disease condition offers incredible opportunities for improved medical care, and the potential for such testing increases almost daily as ever increasing numbers of disease-associated genes and/or mutations are identified. A major hurdle which must be overcome to realize this potential, however, is the high cost of testing. This is particularly true in the case of highly polymorphic genes where the need to test for a large number of variations may make the test procedure appear to be so expensive that routine testing can never be achieved.
Testing for changes in DNA sequence can proceed via complete sequencing of a target nucleic acid molecule, although many persons in the art believe that such testing is too expensive to ever be routine. Changes in DNA sequence can also be detected by a technique called 'single-stranded conformational polymorphism" ( "SSCP") described by Orita et al., Genomics 5: 874-879 (1989), or by a modification thereof referred to a dideoxy-fingerprinting ("ddF") described by Sarkar et al., Genomics 13:
4410443 (1992).
SSCP and ddF both evaluate the pattern of bands created when DNA fragments are electrophoretically separated on a non-denaturing electrophoresis gel. This pattern depends on a combination of the size of the fragments and of the three-dimensional conformation of the undenatured fragments. Thus, the pattern cannot be used for sequencing, because the theoretical spacing of the fragment bands is not equal.
This application relates to a particular test which can be useful as part of a testing protocol for the detection and characterization of human irnmunodeficiency virus (HIV).
SUMMARY OF THE INVENTION
The method of the invention provides a method for obtaining information about the allelic type of a sample of genetic material derived from an HIV-infected sample. A test is performed in which the sequence is determined in the 3'-direction for all four bases. 'This test will identify substantially all of the samples in which the sequence of the sample is determined in both the 3' and 5-direction for all four bases.
To perform the method of the invention, reagents suitable for the tests are suitably packaged as a kit. The kit contains reagents for performing a four-base sequence determination on one or both strands of the target DNA. One-stranded sequence determination could be performed all in the 3'-direction, all in the 5'-direction, or as a S combination of the two strands.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows a schematic representation of the invention.
DETAILED DESCRIPTION OF THE INVENTION
While the terminology used in this application is standard within the art, the following definitions of certain terms are provided to assure clarity.
"Allele" refers to a specific version of a nucleotide sequence at a polymorphic genetic locus.
BACKGROUND OF THE INVENTION
Genetic testing to determine the presence of or a susceptibility to a disease condition offers incredible opportunities for improved medical care, and the potential for such testing increases almost daily as ever increasing numbers of disease-associated genes and/or mutations are identified. A major hurdle which must be overcome to realize this potential, however, is the high cost of testing. This is particularly true in the case of highly polymorphic genes where the need to test for a large number of variations may make the test procedure appear to be so expensive that routine testing can never be achieved.
Testing for changes in DNA sequence can proceed via complete sequencing of a target nucleic acid molecule, although many persons in the art believe that such testing is too expensive to ever be routine. Changes in DNA sequence can also be detected by a technique called 'single-stranded conformational polymorphism" ( "SSCP") described by Orita et al., Genomics 5: 874-879 (1989), or by a modification thereof referred to a dideoxy-fingerprinting ("ddF") described by Sarkar et al., Genomics 13:
4410443 (1992).
SSCP and ddF both evaluate the pattern of bands created when DNA fragments are electrophoretically separated on a non-denaturing electrophoresis gel. This pattern depends on a combination of the size of the fragments and of the three-dimensional conformation of the undenatured fragments. Thus, the pattern cannot be used for sequencing, because the theoretical spacing of the fragment bands is not equal.
This application relates to a particular test which can be useful as part of a testing protocol for the detection and characterization of human irnmunodeficiency virus (HIV).
SUMMARY OF THE INVENTION
The method of the invention provides a method for obtaining information about the allelic type of a sample of genetic material derived from an HIV-infected sample. A test is performed in which the sequence is determined in the 3'-direction for all four bases. 'This test will identify substantially all of the samples in which the sequence of the sample is determined in both the 3' and 5-direction for all four bases.
To perform the method of the invention, reagents suitable for the tests are suitably packaged as a kit. The kit contains reagents for performing a four-base sequence determination on one or both strands of the target DNA. One-stranded sequence determination could be performed all in the 3'-direction, all in the 5'-direction, or as a S combination of the two strands.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows a schematic representation of the invention.
DETAILED DESCRIPTION OF THE INVENTION
While the terminology used in this application is standard within the art, the following definitions of certain terms are provided to assure clarity.
"Allele" refers to a specific version of a nucleotide sequence at a polymorphic genetic locus.
2. "Polymorphic site" means a given nucleotide location in a genetic locus which is variable within a population.
3. "Gene" or "Genetic locus" means a specific nucleotide sequence within a given genome.
4. The "location" or "position" of a nucleotide in a genetic locus means the number assigned to the nucleotide in the gene, generally taken from the cDNA sequence or the genomic sequence of the gene.
5. The nucleotides Adenine, Cytosine, Guanine and Thymine are sometimes represented by their designations of A, C, G or T, respectively. Dideoxynucleotides which are used as chain terminators are abbreviated as ddA, ddC, ddG and ddT.
While it has long been apparent to persons skilled in the art that knowledge of the identity of the base at a particular location within a polymorphic genetic locus may be sufficient to determine the allelic type of that locus, this knowledge has not led to any modification of sequencing procedures. Rather, the knowledge has driven development of techniques such as allele-specific hybridization assays, and allele-specific ligation assays.
Despite the failure of the art to recognize the possibility, however, it is not always necessary to determine the sequence of all four nucleotides of a polymorphic genetic locus in order to determine which allele is present in a specific patient sample. As disclosed generally in International Patent Publication No. WO 97/23650, certain alleles of a genetic locus may be distinguishable on the basis of identification of the location of less than four, and often only one nucleotide. This finding allows the development of the present method for improved allele identification within the highly polymorphic HIV genome.
Traditionally, if sequencing were going to be used to evaluate the allelic type of a polymorphic gene, four dideoxy nucleotide "sequencing" reactions of the type described by Sanger et al. (Proc. Natl. Acad. Sci. USA 74: 5463-5467 (1977)) would be run on the sample concurrently, and the products of the four reactions would then be analyzed by polyacrylamide gel electrophoresis. (see Chp 7.6, Current Protocols in Molecular Biology, Eds. Ausubel, F.M. et al, (John Wiley & Sons; 1995)) In this well-known technique, each of the four sequencing reactions generates a plurality of primer extension products, all of which end with a specific type of dideoxynucleotide. Each lane on the electrophoresis gel thus reflects the positions of one type of base in the extension product, but does not reveal the order and type of nucleotides intervening between the bases of this specific type. The information provided by the four lanes is therefore combined in known sequencing procedures to arnve at a composite picture of the sequence as a whole.
In the method of the invention the sequence of a good portion of the diagnostically relevant protease and reverse transcriptase genes is obtained in three steps:
1 ) cDNA is generated from the RNA present in the sample, and amplified, preferably across a region extending from 6 codons before the protease up to codon 335 of the reverse transcriptase of HIV-1 (the primer regions are not included in this range). 2) Sequencing reactions are performed. 3) Finally, the sequencing ladders are analyzed, preferably using the OpenGeneTM System: the MicroGene ClipperTM or Long-Read TowerTM DNA Sequencers, GeneObjectsTM and GeneLibrarianTM Software.
Fig. 1 shows one embodiment of the method of the invention schematically.
As shown, an RNA sample is obtained and treated by reverse transcriptase-PCR
(RT-PCR) to produce an amplicon of approximately 1.3 kbase pairs spanning the protease and reverse transcriptase genes of the HIV genome from a target cell. This reaction can be performed using, for example, the TITANTM One-Tube RT-PCR system from Boehringer Mannheim (Cat. No. 1 855 476 or 1 882 382) using the following primers:
forward primer set:
AAGCAGGAGC CGATAGACAA GG SEQ ID No. 1 AAGCAGGAGC HGAWAGACAR GG SEQ ID No. 2 CAGCAGGAAC CGAGGGACAA GG SEQ ID No. 3 reverse primer set:
CTAYTARGTC TTTTGWTGGG TCATA SEQ ID No. 4 GCTATTAAGT CTTTTGATGG GTCA SEQ ID No. 5 This amplicon is then combined with a master sequencing mixture containing buffer (260 mM Tris-HCL, pH 8.3; 32.5 mM MgCl2 at 25 °C) and a polymerase enzyme such as Taq FS (Perkin Elmer/Applied Biosystems Cat No. 402070) This polyrnerase has a high rate of incorporartion of dideoxynucleotide relateive to the incorporation rate of, for example, conventional Taq polymerase. This mixture is used as stock in the subsequent reactions.
The sequence reaction is performed on the protease gene using the following primers:
forward primers:
GAGCCRATAG ACAAGGAAYT RTAT SEQ ID No. 6 GAGMCGATAG ACAAGGRVCT GTAT SEQ ID No. 7 reverse primers:
ACTTTTGGGC CATCCATTCC T SEQ ID No. 8 Other forward primers which could be used at this step include:
ID
No.
GAGCCGATAG ACAAGGAAGT ATATCC SEQ ID No. 10 GAGCCGATAG ACAAGGAAAT ATATCC SEQ ID No. 11 GAGCCGATAG ACAAGGAACT GTATCC SEQ ID No. 12 GAGCCGATAG ACAAGGAAGT GTATCC SEQ ID No. 13 GAGCCGATAG ACAAGGAAAT GTATCC SEQ ID No. 14 GAGCCGATAG ACAAGGGACT GTATCC SEQ ID No. 15 GAGCCGATAG ACAAGGACCT GTATCC SEQ ID No. 16 GAGCCGATAG ACAAGGGCCT GTATCC SEQ ID No. 17 GAGCCGATAG ACAAGGAGCT GTATCC SEQ ID No. 18 GAGCCGATAG ACAAGGGGCT GTATCC SEQ ID No. 19 For the reverse transcriptase gene, three sets of primers are used as follows:
RT 1 Primers forward:
GTTAAACAAT GGCCATTGAC AGAAGA SEQ ID No. 20 reverse:
GGAATATTGC TGGTGATCCT TTCC SEQ ID No. 21 alternate forward:
GTTAAACAAT GGCCATTGAC AG SEQ ID No. 22 RT2 Primers forward:
GAAGTATACT GCATTTACCA TACCTAG SEQ ID No. 23 GAAGTATACT GCATTTACTA TACCTAG SEQ ID No. 24 AAAGTATACT GCATTCACCA TACCTAG SEQ ID No. 25 GAAATATACC GCATTTACCA TAYCTAG SEQ ID No. 26 reverse:
TCTGTATGTC ATTGACAGTC CAGC SEQ ID No. 27 alternate reverse:
TCTGTATATC ATTGACAGTC CAGT SEQ ID No. 28 TCTGTATATC ATTGACAGTC CAGC SEQ ID No. 29 TTCTGTATGT CATTGACAGT CCAGC SEQ ID No. 30 P2 Primers forward:
TTCCCTCAGA TCACTCTTTG G SEQ ID No. 31 TTCCCTCAAA TCACTCTTTG G SEQ ID No. 32 reverse:
ACTTTTGGGC CATCCATTCC T SEQ ID No. 33 The P2 forward primers are nested within the PR forward primers to sequence samples which do not sequence with the PR primers. When a sequencing device is employed which is capable of detecting and distinguishing two different fluorescent dyes (such as, for example, the Visible Genetics Inc. MicroGene Clipper or Long-Read Tower sequencers), both the forwaid and reverse primers are preferably each labeled with one of the two dyes. Forward and reverse sequencing fragments are then generated by thermally cycling the sample through multiple thermal cycles in the presence of either ddA, ddT, ddC and ddG. Analysis of the sequencing fragments produced using gel electrophoresis will allow the determination of the positions of all 4 bases.
Finally, if the intermediate test fails to provide unambiguous identification of the DNA type, sequencing of both strands may be performed. Again, the same sequencing primers identified above are used. Forward and reverse sequencing fragments can be produced in a single reaction using distinctively labeled forward and reverse primers, or in separate reactions depending on the nature of the detection system being employed.
Reagents suitable for practicing the method of the invention are suitably packaged in kit form. Thus, the invention provides a kit for analyzing the genetic type of an HIV-1 gene in a sample comprising: a kit for performing four base sequencing on HIV-1 comprising a plurality of A, C, G and T terminations mixtures, each of said termination mixtures including one of a plurality of primer pairs, each pair flanking a different region of the HIV-1 genome, the pairs together flanking substantially all of the protease and reverse transcriptase genes, and at least one member of each pair being labeled with a detectable label. Additional subkits for performing four base sequencing may be included when intermediate and final assays on one strand and both strands are desired.
As used herein, the term "termination mixture" refers to a mixture containing a mixture of the four deoxunucleotide triphosphates (dATP, dCTP, dGTP, and dTTP), one species of chain terminating dideoxynucleotide (ddATP, ddCTP, ddGTP or ddTTP) and the appropriate sequencing primers.
The subkit for performing A and T sequencing on HIV-1 may also be provided separately for performing the initial determination of only the A and T
nucleotides. A
preferred kit of this type, whether provided separately or as part of a kit for performing a hierarchical assay has primer pairs in which each primer is labeled with a different an spectroscopically distinguishable fluorescent dye, such as Cy5.0 and Cy5.5 and includes only one of the two possible types of termination mixtures, for example just the T
termination mixture.
The following examples are included to illustrate aspects of the instant invention and are not intended to limit the invention in any way.
While it has long been apparent to persons skilled in the art that knowledge of the identity of the base at a particular location within a polymorphic genetic locus may be sufficient to determine the allelic type of that locus, this knowledge has not led to any modification of sequencing procedures. Rather, the knowledge has driven development of techniques such as allele-specific hybridization assays, and allele-specific ligation assays.
Despite the failure of the art to recognize the possibility, however, it is not always necessary to determine the sequence of all four nucleotides of a polymorphic genetic locus in order to determine which allele is present in a specific patient sample. As disclosed generally in International Patent Publication No. WO 97/23650, certain alleles of a genetic locus may be distinguishable on the basis of identification of the location of less than four, and often only one nucleotide. This finding allows the development of the present method for improved allele identification within the highly polymorphic HIV genome.
Traditionally, if sequencing were going to be used to evaluate the allelic type of a polymorphic gene, four dideoxy nucleotide "sequencing" reactions of the type described by Sanger et al. (Proc. Natl. Acad. Sci. USA 74: 5463-5467 (1977)) would be run on the sample concurrently, and the products of the four reactions would then be analyzed by polyacrylamide gel electrophoresis. (see Chp 7.6, Current Protocols in Molecular Biology, Eds. Ausubel, F.M. et al, (John Wiley & Sons; 1995)) In this well-known technique, each of the four sequencing reactions generates a plurality of primer extension products, all of which end with a specific type of dideoxynucleotide. Each lane on the electrophoresis gel thus reflects the positions of one type of base in the extension product, but does not reveal the order and type of nucleotides intervening between the bases of this specific type. The information provided by the four lanes is therefore combined in known sequencing procedures to arnve at a composite picture of the sequence as a whole.
In the method of the invention the sequence of a good portion of the diagnostically relevant protease and reverse transcriptase genes is obtained in three steps:
1 ) cDNA is generated from the RNA present in the sample, and amplified, preferably across a region extending from 6 codons before the protease up to codon 335 of the reverse transcriptase of HIV-1 (the primer regions are not included in this range). 2) Sequencing reactions are performed. 3) Finally, the sequencing ladders are analyzed, preferably using the OpenGeneTM System: the MicroGene ClipperTM or Long-Read TowerTM DNA Sequencers, GeneObjectsTM and GeneLibrarianTM Software.
Fig. 1 shows one embodiment of the method of the invention schematically.
As shown, an RNA sample is obtained and treated by reverse transcriptase-PCR
(RT-PCR) to produce an amplicon of approximately 1.3 kbase pairs spanning the protease and reverse transcriptase genes of the HIV genome from a target cell. This reaction can be performed using, for example, the TITANTM One-Tube RT-PCR system from Boehringer Mannheim (Cat. No. 1 855 476 or 1 882 382) using the following primers:
forward primer set:
AAGCAGGAGC CGATAGACAA GG SEQ ID No. 1 AAGCAGGAGC HGAWAGACAR GG SEQ ID No. 2 CAGCAGGAAC CGAGGGACAA GG SEQ ID No. 3 reverse primer set:
CTAYTARGTC TTTTGWTGGG TCATA SEQ ID No. 4 GCTATTAAGT CTTTTGATGG GTCA SEQ ID No. 5 This amplicon is then combined with a master sequencing mixture containing buffer (260 mM Tris-HCL, pH 8.3; 32.5 mM MgCl2 at 25 °C) and a polymerase enzyme such as Taq FS (Perkin Elmer/Applied Biosystems Cat No. 402070) This polyrnerase has a high rate of incorporartion of dideoxynucleotide relateive to the incorporation rate of, for example, conventional Taq polymerase. This mixture is used as stock in the subsequent reactions.
The sequence reaction is performed on the protease gene using the following primers:
forward primers:
GAGCCRATAG ACAAGGAAYT RTAT SEQ ID No. 6 GAGMCGATAG ACAAGGRVCT GTAT SEQ ID No. 7 reverse primers:
ACTTTTGGGC CATCCATTCC T SEQ ID No. 8 Other forward primers which could be used at this step include:
ID
No.
GAGCCGATAG ACAAGGAAGT ATATCC SEQ ID No. 10 GAGCCGATAG ACAAGGAAAT ATATCC SEQ ID No. 11 GAGCCGATAG ACAAGGAACT GTATCC SEQ ID No. 12 GAGCCGATAG ACAAGGAAGT GTATCC SEQ ID No. 13 GAGCCGATAG ACAAGGAAAT GTATCC SEQ ID No. 14 GAGCCGATAG ACAAGGGACT GTATCC SEQ ID No. 15 GAGCCGATAG ACAAGGACCT GTATCC SEQ ID No. 16 GAGCCGATAG ACAAGGGCCT GTATCC SEQ ID No. 17 GAGCCGATAG ACAAGGAGCT GTATCC SEQ ID No. 18 GAGCCGATAG ACAAGGGGCT GTATCC SEQ ID No. 19 For the reverse transcriptase gene, three sets of primers are used as follows:
RT 1 Primers forward:
GTTAAACAAT GGCCATTGAC AGAAGA SEQ ID No. 20 reverse:
GGAATATTGC TGGTGATCCT TTCC SEQ ID No. 21 alternate forward:
GTTAAACAAT GGCCATTGAC AG SEQ ID No. 22 RT2 Primers forward:
GAAGTATACT GCATTTACCA TACCTAG SEQ ID No. 23 GAAGTATACT GCATTTACTA TACCTAG SEQ ID No. 24 AAAGTATACT GCATTCACCA TACCTAG SEQ ID No. 25 GAAATATACC GCATTTACCA TAYCTAG SEQ ID No. 26 reverse:
TCTGTATGTC ATTGACAGTC CAGC SEQ ID No. 27 alternate reverse:
TCTGTATATC ATTGACAGTC CAGT SEQ ID No. 28 TCTGTATATC ATTGACAGTC CAGC SEQ ID No. 29 TTCTGTATGT CATTGACAGT CCAGC SEQ ID No. 30 P2 Primers forward:
TTCCCTCAGA TCACTCTTTG G SEQ ID No. 31 TTCCCTCAAA TCACTCTTTG G SEQ ID No. 32 reverse:
ACTTTTGGGC CATCCATTCC T SEQ ID No. 33 The P2 forward primers are nested within the PR forward primers to sequence samples which do not sequence with the PR primers. When a sequencing device is employed which is capable of detecting and distinguishing two different fluorescent dyes (such as, for example, the Visible Genetics Inc. MicroGene Clipper or Long-Read Tower sequencers), both the forwaid and reverse primers are preferably each labeled with one of the two dyes. Forward and reverse sequencing fragments are then generated by thermally cycling the sample through multiple thermal cycles in the presence of either ddA, ddT, ddC and ddG. Analysis of the sequencing fragments produced using gel electrophoresis will allow the determination of the positions of all 4 bases.
Finally, if the intermediate test fails to provide unambiguous identification of the DNA type, sequencing of both strands may be performed. Again, the same sequencing primers identified above are used. Forward and reverse sequencing fragments can be produced in a single reaction using distinctively labeled forward and reverse primers, or in separate reactions depending on the nature of the detection system being employed.
Reagents suitable for practicing the method of the invention are suitably packaged in kit form. Thus, the invention provides a kit for analyzing the genetic type of an HIV-1 gene in a sample comprising: a kit for performing four base sequencing on HIV-1 comprising a plurality of A, C, G and T terminations mixtures, each of said termination mixtures including one of a plurality of primer pairs, each pair flanking a different region of the HIV-1 genome, the pairs together flanking substantially all of the protease and reverse transcriptase genes, and at least one member of each pair being labeled with a detectable label. Additional subkits for performing four base sequencing may be included when intermediate and final assays on one strand and both strands are desired.
As used herein, the term "termination mixture" refers to a mixture containing a mixture of the four deoxunucleotide triphosphates (dATP, dCTP, dGTP, and dTTP), one species of chain terminating dideoxynucleotide (ddATP, ddCTP, ddGTP or ddTTP) and the appropriate sequencing primers.
The subkit for performing A and T sequencing on HIV-1 may also be provided separately for performing the initial determination of only the A and T
nucleotides. A
preferred kit of this type, whether provided separately or as part of a kit for performing a hierarchical assay has primer pairs in which each primer is labeled with a different an spectroscopically distinguishable fluorescent dye, such as Cy5.0 and Cy5.5 and includes only one of the two possible types of termination mixtures, for example just the T
termination mixture.
The following examples are included to illustrate aspects of the instant invention and are not intended to limit the invention in any way.
The RT-PCR is done on the HIV-1 RNA using a blend of enzymes forming RT-PCR Master Mixes described below to conduct six RT-PCT reactions. This RT-PCR
is done on the RNA preparation obtained using the QIAmp Viral RNA kit from Qiagen. It can also be done on the RNA extract for the NucliSenseTM (formerly known as NASBA) HIV Viral Load from Organon Teknica.
All the reagents, tubes, tips, and other material needs to be RNase-free.
The recipe is made for 8 reactions (one strip of 8 tubes), including 10%
extra. Thaw the RNA sample from the Amplicor HIV Monitor Test and keep on ice. This is the material obtained at step 14 of the section B "Specimen Preparation". If using RNA
prepared for the NucliSense Assay, proceed the same way: thaw it and keep it on ice.
Take a 0.2 ml sterile, RNase-free, centrifuge tube, RNase-free, and prepare the RT-PCR Master Mix I (enough for 6 tubes, i.e. 6 samples) by adding the following ingredient in the order listed:
RT-PCR MASTER MIX I
is done on the RNA preparation obtained using the QIAmp Viral RNA kit from Qiagen. It can also be done on the RNA extract for the NucliSenseTM (formerly known as NASBA) HIV Viral Load from Organon Teknica.
All the reagents, tubes, tips, and other material needs to be RNase-free.
The recipe is made for 8 reactions (one strip of 8 tubes), including 10%
extra. Thaw the RNA sample from the Amplicor HIV Monitor Test and keep on ice. This is the material obtained at step 14 of the section B "Specimen Preparation". If using RNA
prepared for the NucliSense Assay, proceed the same way: thaw it and keep it on ice.
Take a 0.2 ml sterile, RNase-free, centrifuge tube, RNase-free, and prepare the RT-PCR Master Mix I (enough for 6 tubes, i.e. 6 samples) by adding the following ingredient in the order listed:
RT-PCR MASTER MIX I
7 ~l of 80 mM DTT
10.5 ~,l of RNase-free dNTP at 10 mM each dNTP
21 ~,1 of forward PCR primer at 28 ~.M.
21 ~,1 of reverse PCR primer at 28 ~,M
3.5 ~,1 of Rnase-inhibitor from Roche Molecular Biochemicals, catalog # 799 025 (10,000 U) Take a 0.2 ml sterile, RNase-free, centrifuge tube, RNase-free, and prepare the RT-PCR Master Mix II (enough for 6 tubes) by adding the following ingredient in the order listed:
RT-PCR MASTER MIX II
70 ~1 of 4x RT-PCR Buffer (280 mM Tris Hcl, 9.2 mM MgCl2, 60 mM (NH4)zS04, 100 ~,g/ml Acetylated BSA from Life Tech, CA, pH 8.60 at 25°C) 3.5 ~1 of RNase Inhibitor at40 U/~,l 7 ~.1 of Superscript II
_7_ 8.75 ~.l of Expand High Fidelity Enzyme System Enzyme Mix from Roche Molecular Biochemicals, catalog # 17333 818 8.75 ~.1 of AmpliTaq from Roche Molecular Systems.
Take one strip of 6 thin wall tubes. Add 9 ~l of MASTER MIX I in each tube.
Add 17 ~,1 of sample (RNA) to each tube. You may want to add a negative control per experiment. Heat the RNA sample at 90°C for 2 min. using the program below:, cool at 50°C and add 14 ~,1 of the MASTER MIX II in each tube (step 3 of the program below). Be careful not to cross contaminate your samples.
Start the RT-PCR. Use the heated lid. When using the MJ-Plates, indicate that tubes are used when asked by the PTC-200. The following is the programming for the PTC-200:
Calculated 1= 90.0° for ever 2= 90.0° for 2:00 3= 50.0° for 1:00:00 4= 94.0° for 2:00 5= 1.0°/s to 94.0°
6= 94.0° for 0:30 7= 1.0°/s to 57.0°
8= 57.0° for 0:30 9= 1.0°/s to 68.0°
10.5 ~,l of RNase-free dNTP at 10 mM each dNTP
21 ~,1 of forward PCR primer at 28 ~.M.
21 ~,1 of reverse PCR primer at 28 ~,M
3.5 ~,1 of Rnase-inhibitor from Roche Molecular Biochemicals, catalog # 799 025 (10,000 U) Take a 0.2 ml sterile, RNase-free, centrifuge tube, RNase-free, and prepare the RT-PCR Master Mix II (enough for 6 tubes) by adding the following ingredient in the order listed:
RT-PCR MASTER MIX II
70 ~1 of 4x RT-PCR Buffer (280 mM Tris Hcl, 9.2 mM MgCl2, 60 mM (NH4)zS04, 100 ~,g/ml Acetylated BSA from Life Tech, CA, pH 8.60 at 25°C) 3.5 ~1 of RNase Inhibitor at40 U/~,l 7 ~.1 of Superscript II
_7_ 8.75 ~.l of Expand High Fidelity Enzyme System Enzyme Mix from Roche Molecular Biochemicals, catalog # 17333 818 8.75 ~.1 of AmpliTaq from Roche Molecular Systems.
Take one strip of 6 thin wall tubes. Add 9 ~l of MASTER MIX I in each tube.
Add 17 ~,1 of sample (RNA) to each tube. You may want to add a negative control per experiment. Heat the RNA sample at 90°C for 2 min. using the program below:, cool at 50°C and add 14 ~,1 of the MASTER MIX II in each tube (step 3 of the program below). Be careful not to cross contaminate your samples.
Start the RT-PCR. Use the heated lid. When using the MJ-Plates, indicate that tubes are used when asked by the PTC-200. The following is the programming for the PTC-200:
Calculated 1= 90.0° for ever 2= 90.0° for 2:00 3= 50.0° for 1:00:00 4= 94.0° for 2:00 5= 1.0°/s to 94.0°
6= 94.0° for 0:30 7= 1.0°/s to 57.0°
8= 57.0° for 0:30 9= 1.0°/s to 68.0°
10= 68.0° for 2:00 11=Goto 5, 19 times 12= 1.0°/s to 94.0°
13= 94.0° for 0:30 14= 1.0°/s to 60.0°
15= 60.0° for 0:30 16= 1.0°/s to 68.0°
17= 68.0° for 2:30 18=Goto 12, 16 times 19= 68.0° for 7:00 20= 4.0° for ever _g_ 21=End To determine the sequence of amplicon, 7 ~l of each terminator mix (16 when using a two dye instrument) are combined with a 5 ul of a master mix as follows:
MASTER MIX (two-dye system) for 6 tubes, i.e. for 6 samples:
120 ~,1 of buffer (260 mM Tris-HCI, pH 8.3 at 25°C, 32.5 mM MgCl2) 475 ~,l of sterile water 22.5 ~,l enzyme blend of AmpliTaq FS from Roche Molecular Systems 15 U/~,1 and U/~,l pyrophosphotase S ~.l of the PCR product from Example 3 per tube.
The two mixtures are mixed gently with a pipette tip, and the thermocylcing reaction is started. The following is the programming for the PTC-200:
Calculated 1= 94.0° for 5:00 2= 1.0°/s to 94.0°
3= 94.0° for 0:20 4= 1.0°/s to 56.0°
5= 56.0° for 0:20 6= 1.0°/s to 70.0°
7= 70.0° for 1:30 8=Goto 2, 29 times 9= 70.0° for 5:00 10= 4.0° for ever 11=End Termination mixes for two dye systems Protease A-Mix: 1.07 ~M ddATP; 643 ~M dATP; 643 ~M dCTP; 643 ~.M dGTP; 643 ~M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers; 1 mM Tris-HCI, pH
8.0 at 25°C, 0.1 mM EDTA.
C-Mix: 2.14 ~.M ddCTP; 643 ~.M dATP; 643 ~,M dCTP; 643 wM dGTP; 643 ~M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers; 1 mM Tris-HCI, pH
8.0 at 25°C, 0.1 mM EDTA.
G-Mix: 2.14 ~,M ddGTP; 643 ~.M dATP; 643 ~,M dCTP; 643 ~,M dGTP; 643 ~M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers; 1 mM Tris-HCI, pH
8.0 at 25°C, 0.1 mM EDTA.
T-Mix: 2.14 ~M ddTTP; 643 wM dATP; 643 ~M dCTP; 643 ~M dGTP; 643 ~,M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers; 1 mM Tris-HCI, pH
8.0 at 25°C, 0.1 mM EDTA.
Both primers are labeled, for example with Cy5.0 and Cy5.5, respectively.
First RT region A-Mix: 1.07 wM ddATP; 643 ~,M dATP; 643 ~.M dCTP; 643 ~.M dGTP; 643 ~,M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers; 1 mM Tris-HCI, pH
8.0 at 25°C, 0.1 mM EDTA.
C-Mix: 2.14 ~M ddCTP; 643 ~,M dATP; 643 wM dCTP; 643 ~,M dGTP; 643 ~M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers; 1 mM Tris-HCI, pH
8.0 at 25°C, 0.1 mM EDTA.
G-Mix: 2.14 ~,M ddGTP; 643 ~M dATP; 643 ~,M dCTP; 643 ~M dGTP; 643 ~,M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers; 1 mM Tris-HCI, pH
8.0 at 25°C, 0.1 mM EDTA.
T-Mix: 2.14 ~.M ddTTP; 643 ~,M dATP; 643 ~,M dCTP; 643 ~M dGTP; 643 ~.M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers Both primers are labeled, for example with Cy5.0 and Cy5.5, respectively.
Second reverse transcriptase region A-Mix: 1.07 ~,M ddATP; 643 ~M dATP; 643 ~,M dCTP; 643 ~,M dGTP; 643 ~M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers C-Mix: 2.14 ~M ddCTP; 643 ~,M dATP; 643 ~M dCTP; 643 ~M dGTP; 643 ~M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers G-Mix: 2.14 ~,M ddGTP; 643 ~M dATP; 643 ~M dCTP; 643 ~.M dGTP; 643 ~M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers T-Mix: 2.14 ~M ddTTP; 643 ~M dATP; 643 ~M dCTP; 643 ~M dGTP; 643 ~,M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers Both primers are labeled, for example with Cy5.0 and Cy5.5, respectively.
P2 protease region A-Mix: 1.07 ~,M ddATP; 643 ~M dATP; 643 ~,M dCTP; 643 ~,M dGTP; 643 ~.M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers C-Mix: 2.14 ~.M ddCTP; 643 ~M dATP; 643 wM dCTP; 643 ~M dGTP; 643 ~M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers G-Mix: 2.14 ~,M ddGTP; 643 ~.M dATP; 643 ~,M dCTP; 643 ~,M dGTP; 643 ~M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers T-Mix: 2.14 ~,M ddTTP; 643 ~.M dATP; 643 ~M dCTP; 643 ~.M dGTP; 643 ~,M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers Both primers are labeled, for example with Cy5.0 and Cy5.5, respectively.
i CA 02317789 2001-02-21 SEQUENCE LISTING
Applicant: VISIBLE GENETICS INC.
Title: Method and Kit for Evaluation of HIV Mutations Number of Sequences:33 Correspondence Address: Ernst & Young Tower 222 Bay Street, Suite 1800 P.O. Box 197, T.D. Centre Toronto, Ontario Canada M5K 1H6 Computer-Readable Form Computer: IBM Compatiable Operating System: MS-DOS
Current Application No:2,317,789 Current Filing Date: 2000-09-14 Earlier Application No: 09/418,720 Earlier Filing Date:1999-10-15 PATENT AGENT INFORMATION:
NAME: DONAHUE ERNST & YOUNG
File Reference:2393-003331 INFORMATION FOR FOR SEQ.ID N0: 1 SEQUENCE CHARACTERISTICS:
LENGTH: 22 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: amplification primer for HIV
SEQUENCE DESCRIPTION: SEQ ID N0. 1 aagcaggagc cgatagacaa gg 22 INFORMATION FOR SEQ ID N0: 2 SEQUENCE CHARACTERISTICS:
LENGTH: 22 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: amplification primer for HIV
SEQUENCE DESCRIPTION: SEQ ID N0. 2 aagcaggagc hgawagacar gg 22 INFORMATION FOR SEQUENCE ID N0. 3 SEQUENCE CHARACTERISTICS:
LENGTH: 22 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE
OTHER INFORMATION: amplification primer for HIV
SEQUENCE DESCRIPTION: SEQ ID N0. 3 cagcaggaac cgagggacaa gg 22 INFORMATION FOR SEQUENCE ID N0. 4 SEQUENCE CHARACTERISTICS:
LENGTH: 25 TYPE: DNA
ORIGNIAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: amplification primer for HIV
SEQUENCE DESCRIPTION: SEQ ID N0. 4 ctaytargtc ttttgwtggg tcata 25 INFORMATION FOR SEQ ID N0.5 SEQUENCE CHARACTERISTICS:
LENGTH: 25 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: amplification primer for HIV
SEQUENCE DESCRIPTION: SEQ ID N0: 5 gctattaagt cttttgatgg gtca 24 INFORMATION FOR SEQ ID N0: 6 SEQUENCE CHARACTERISTICS:
LENGTH: 24 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION: SEQ ID N0: 6 gagccratag acaaggaayt rtat 24 INFORMATION FOR SEQ ID N0:7 SEQUENCE CHARACTERISTICS:
LENGTH: 24 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION: SEQ ID N0:7 gagmcgatag acaaggrvct gtat 24 INFORMATION FOR SEQ ID N0: 8 SEQUENCE CHARACTERISTICS:
LENGTH: 21 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene ' CA 02317789 2001-02-21 SEQUENCE DESCRIPTION: SEQ ID N0:8 acttttgggc catccattcc t 21 INFORMATION FOR SEQ ID N0: 9 SEQUENCE CHARACTERISTICS:
LENGTH: 26 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION:SEQ ID N0:9 gagccgatag acaaggaact atatcc 26 INFORMATION FOR SEQUENCE ID N0. 10 SEQUENCE CHARACTERISTICS:
LENGTH: 26 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION:SEQ ID N0: 10 gagccgatag acaaggaagt atatcc 26 ' CA 02317789 2001-02-21 INFORMATION FOR SEQ ID N0:11 SEQUENCE CHARACTERISTICS:
LENGTH: 26 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION: SEQ ID N0. 11 gagccgatag acaaggaaat atatcc 26 INFORMATION FOR SEQ ID NO: 12 SEQUENCE CHARACTERISTICS:
LENGTH: 26 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION: SEQ ID N0: 12 gagccgatag acaaggaact gtatcc 26 INFORMATION FOR SEQ ID NO: 13 SEQUENCE CHARACTERISTICS:
LENGTH: 26 TYPE: DNA
' CA 02317789 2001-02-21 ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION: SEQ ID N0: 13 gagccgatag acaaggaagt gtatcc 26 INFORMATION FOR SEQ ID N0: 14 SEQUENCE CHARACTERISTICS:
LENGTH: 26 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION: SEQ ID N0: 14 gagccgatag acaaggaaat gtatcc 26 INFORMATION FOR SEQ ID N0: 15 SEQUENCE CHARACTERISTICS:
LENGTH: 26 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION FOR ID N0: 15 gagccgatag acaagggact gtatcc 26 INFORMATION FOR SEQ ID N0: 16 SEQUENCE CHARACTERISTICS:
LENGTH:26 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION: SEQ ID N0: 16 gagccgatag acaaggacct gtatcc 26 INFORMATION FOR SEQ ID NO: 17 SEQUENCE CHARACTERISTICS:
LENGTH: 26 TYPE: DNA
ORIGNAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION: SEQ ID N0: 17 gagccgatag acaagggcct gtatcc 26 INFORMATION FOR SEQ ID NO: 18 SEQUENCE CHARACTERISTICS:
LENGTH: 26 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION: SEQ ID NO: 18 gagccgatag acaaggagct gtatcc 26 INFORMATION FOR SEQ ID N0: 19 SEQUENCE CHARACTERISTICS:
LENGTH: 26 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION: SEQ ID N0: 19 gagccgatag acaaggggct gtatcc 26 INFORMATION FOR SEQ ID N0: 20 SEQUENCE CHARACTERISTICS:
LENGTH: 26 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: RT1 forward primer SEQUENCE DESCRIPTION:SEQ ID NO: 20 gttaaacaat ggccattgac agaaga 26 INFORMATION FOR SEQ ID N0: 21 SEQUENCE CHARACTERISTICS:
LENGTH: 24 TYPE: DNA
ORIGNAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: RT1 reverse primer SEQUENCE DESCRIPTION:SEQ ID N0: 21 ggaatattgc tggtgatcct ttcc 24 INFORMATION FOR SEQ ID N0: 22 SEQUENCE CHARACTERISTICS:
LENGTH: 22 TYPE: DNA
ORGANISM: Human immunodeficiency virus OTHER INFORMATION: RT1 forward primer SEQUENCE DESCRIPTION:SEQ ID N0: 22 gttaaacaat ggccattgac ag 22 INFORMATION FOR SEQ ID N0: 23 SEQUENCE CHARACTERISTICS:
LENGTH: 27 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: RT2 forward sequencing primer SEQUENCE DESCRIPTION: SEQ ID NO: 23 gaagtatact gcatttacca tacctag 27 INFORMATION FOR SEQ ID N0: 24 SEQUENCE CHARACTERISTICS:
LENGTH: 27 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: RT2 forward pimer SEQUENCE DESCRIPTION: SEQ ID N0: 24 gaagtatact gcatttacta tacctag 27 INFORMATION FOR SEQ ID N0: 25 SEQUENCE CHARACTERISTICS:
LENGTH: 27 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: RT2 forward primer SEQUENCE DESCRIPTION: SEQ ID N0: 25 aaagtatact gcattcacca tacctag 27 INFORMATION FOR SEQ ID N0: 26 SEQUENCE CHARACTERSTICS:
LENGTH: 27 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: RT2 forward primer SEQUENCE DESCRIPTION: SEQ ID N0: 26 gaaatatacc gcatttacca tayctag 27 INFORMATION FOR SEQ ID N0: 27 SEQUENCE CHARACTERISTICS:
LENGTH: 24 TYPE: DNA
ORGANISM: Human immunodeficiency virus OTHER INFORMATION: RT2 reverse primer SEQUENCE DESCRIPTION: SEQ ID N0: 27 tctgtatgtc attgacagtc cagc 24 INFORMATION FOR SEQ ID N0: 28 SEQUENCE CHARACTERISTICS:
LENGTH: 24 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: RT2 reverse primer SEQUENCE DESCRIPTION: SEQ ID N0: 28 tctgtatatc attgacagtc cagt 24 INFORMATION FOR SEQ ID N0: 29 SEQUENCE CHARACTERISTICS:
LENGTH: 24 TYPE: DNA
ORIGNAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: RT2 reverse primer SEQUENCE DESCRIPTION:SEQ ID N0: 29 tctgtatatc attgacagtc cagc 24 INFORMATION FOR SEQ ID N0: 30 SEQUENCE CHARACTERISTICS:
LENGTH: 25 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: RT2 reverse primer SEQUENCE DESCRIPTION: SEQ ID NO: 30 ttctgtatgt cattgacagt ccagc ~ 25 INFORMATION SEQ ID N0: 31 SEQUENCE CHARACTERISTICS:
LENGTH: 21 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: P2 forward primer SEQUENCE DESCRIPTION: SEQ ID N0: 31 ttccctcaga tcactctttg g 21 INFORMATION FOR SEQ ID N0: 32 SEQUENCE CHARACTERISTICS:
LENGTH:21 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMTAION: P2 forward primer SEQUENCE DESCRIPTION: SEQ ID N0: 32 ttccctcaaa tcactctttg g 21 INFORMATION FOR SEQ ID N0: 33 SEQUENCE CHARACTERISTICS:
LENGTH: 21 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: P2 reverse primer SEQUENCE DESCRIPTION: SEQ ID N0: 33 acttttgggc catccattcc t 21
MASTER MIX (two-dye system) for 6 tubes, i.e. for 6 samples:
120 ~,1 of buffer (260 mM Tris-HCI, pH 8.3 at 25°C, 32.5 mM MgCl2) 475 ~,l of sterile water 22.5 ~,l enzyme blend of AmpliTaq FS from Roche Molecular Systems 15 U/~,1 and U/~,l pyrophosphotase S ~.l of the PCR product from Example 3 per tube.
The two mixtures are mixed gently with a pipette tip, and the thermocylcing reaction is started. The following is the programming for the PTC-200:
Calculated 1= 94.0° for 5:00 2= 1.0°/s to 94.0°
3= 94.0° for 0:20 4= 1.0°/s to 56.0°
5= 56.0° for 0:20 6= 1.0°/s to 70.0°
7= 70.0° for 1:30 8=Goto 2, 29 times 9= 70.0° for 5:00 10= 4.0° for ever 11=End Termination mixes for two dye systems Protease A-Mix: 1.07 ~M ddATP; 643 ~M dATP; 643 ~M dCTP; 643 ~.M dGTP; 643 ~M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers; 1 mM Tris-HCI, pH
8.0 at 25°C, 0.1 mM EDTA.
C-Mix: 2.14 ~.M ddCTP; 643 ~.M dATP; 643 ~,M dCTP; 643 wM dGTP; 643 ~M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers; 1 mM Tris-HCI, pH
8.0 at 25°C, 0.1 mM EDTA.
G-Mix: 2.14 ~,M ddGTP; 643 ~.M dATP; 643 ~,M dCTP; 643 ~,M dGTP; 643 ~M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers; 1 mM Tris-HCI, pH
8.0 at 25°C, 0.1 mM EDTA.
T-Mix: 2.14 ~M ddTTP; 643 wM dATP; 643 ~M dCTP; 643 ~M dGTP; 643 ~,M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers; 1 mM Tris-HCI, pH
8.0 at 25°C, 0.1 mM EDTA.
Both primers are labeled, for example with Cy5.0 and Cy5.5, respectively.
First RT region A-Mix: 1.07 wM ddATP; 643 ~,M dATP; 643 ~.M dCTP; 643 ~.M dGTP; 643 ~,M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers; 1 mM Tris-HCI, pH
8.0 at 25°C, 0.1 mM EDTA.
C-Mix: 2.14 ~M ddCTP; 643 ~,M dATP; 643 wM dCTP; 643 ~,M dGTP; 643 ~M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers; 1 mM Tris-HCI, pH
8.0 at 25°C, 0.1 mM EDTA.
G-Mix: 2.14 ~,M ddGTP; 643 ~M dATP; 643 ~,M dCTP; 643 ~M dGTP; 643 ~,M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers; 1 mM Tris-HCI, pH
8.0 at 25°C, 0.1 mM EDTA.
T-Mix: 2.14 ~.M ddTTP; 643 ~,M dATP; 643 ~,M dCTP; 643 ~M dGTP; 643 ~.M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers Both primers are labeled, for example with Cy5.0 and Cy5.5, respectively.
Second reverse transcriptase region A-Mix: 1.07 ~,M ddATP; 643 ~M dATP; 643 ~,M dCTP; 643 ~,M dGTP; 643 ~M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers C-Mix: 2.14 ~M ddCTP; 643 ~,M dATP; 643 ~M dCTP; 643 ~M dGTP; 643 ~M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers G-Mix: 2.14 ~,M ddGTP; 643 ~M dATP; 643 ~M dCTP; 643 ~.M dGTP; 643 ~M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers T-Mix: 2.14 ~M ddTTP; 643 ~M dATP; 643 ~M dCTP; 643 ~M dGTP; 643 ~,M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers Both primers are labeled, for example with Cy5.0 and Cy5.5, respectively.
P2 protease region A-Mix: 1.07 ~,M ddATP; 643 ~M dATP; 643 ~,M dCTP; 643 ~,M dGTP; 643 ~.M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers C-Mix: 2.14 ~.M ddCTP; 643 ~M dATP; 643 wM dCTP; 643 ~M dGTP; 643 ~M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers G-Mix: 2.14 ~,M ddGTP; 643 ~.M dATP; 643 ~,M dCTP; 643 ~,M dGTP; 643 ~M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers T-Mix: 2.14 ~,M ddTTP; 643 ~.M dATP; 643 ~M dCTP; 643 ~.M dGTP; 643 ~,M dTTP;
330 nM total of forward primers and 330 nM total of reverse primers Both primers are labeled, for example with Cy5.0 and Cy5.5, respectively.
i CA 02317789 2001-02-21 SEQUENCE LISTING
Applicant: VISIBLE GENETICS INC.
Title: Method and Kit for Evaluation of HIV Mutations Number of Sequences:33 Correspondence Address: Ernst & Young Tower 222 Bay Street, Suite 1800 P.O. Box 197, T.D. Centre Toronto, Ontario Canada M5K 1H6 Computer-Readable Form Computer: IBM Compatiable Operating System: MS-DOS
Current Application No:2,317,789 Current Filing Date: 2000-09-14 Earlier Application No: 09/418,720 Earlier Filing Date:1999-10-15 PATENT AGENT INFORMATION:
NAME: DONAHUE ERNST & YOUNG
File Reference:2393-003331 INFORMATION FOR FOR SEQ.ID N0: 1 SEQUENCE CHARACTERISTICS:
LENGTH: 22 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: amplification primer for HIV
SEQUENCE DESCRIPTION: SEQ ID N0. 1 aagcaggagc cgatagacaa gg 22 INFORMATION FOR SEQ ID N0: 2 SEQUENCE CHARACTERISTICS:
LENGTH: 22 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: amplification primer for HIV
SEQUENCE DESCRIPTION: SEQ ID N0. 2 aagcaggagc hgawagacar gg 22 INFORMATION FOR SEQUENCE ID N0. 3 SEQUENCE CHARACTERISTICS:
LENGTH: 22 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE
OTHER INFORMATION: amplification primer for HIV
SEQUENCE DESCRIPTION: SEQ ID N0. 3 cagcaggaac cgagggacaa gg 22 INFORMATION FOR SEQUENCE ID N0. 4 SEQUENCE CHARACTERISTICS:
LENGTH: 25 TYPE: DNA
ORIGNIAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: amplification primer for HIV
SEQUENCE DESCRIPTION: SEQ ID N0. 4 ctaytargtc ttttgwtggg tcata 25 INFORMATION FOR SEQ ID N0.5 SEQUENCE CHARACTERISTICS:
LENGTH: 25 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: amplification primer for HIV
SEQUENCE DESCRIPTION: SEQ ID N0: 5 gctattaagt cttttgatgg gtca 24 INFORMATION FOR SEQ ID N0: 6 SEQUENCE CHARACTERISTICS:
LENGTH: 24 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION: SEQ ID N0: 6 gagccratag acaaggaayt rtat 24 INFORMATION FOR SEQ ID N0:7 SEQUENCE CHARACTERISTICS:
LENGTH: 24 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION: SEQ ID N0:7 gagmcgatag acaaggrvct gtat 24 INFORMATION FOR SEQ ID N0: 8 SEQUENCE CHARACTERISTICS:
LENGTH: 21 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene ' CA 02317789 2001-02-21 SEQUENCE DESCRIPTION: SEQ ID N0:8 acttttgggc catccattcc t 21 INFORMATION FOR SEQ ID N0: 9 SEQUENCE CHARACTERISTICS:
LENGTH: 26 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION:SEQ ID N0:9 gagccgatag acaaggaact atatcc 26 INFORMATION FOR SEQUENCE ID N0. 10 SEQUENCE CHARACTERISTICS:
LENGTH: 26 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION:SEQ ID N0: 10 gagccgatag acaaggaagt atatcc 26 ' CA 02317789 2001-02-21 INFORMATION FOR SEQ ID N0:11 SEQUENCE CHARACTERISTICS:
LENGTH: 26 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION: SEQ ID N0. 11 gagccgatag acaaggaaat atatcc 26 INFORMATION FOR SEQ ID NO: 12 SEQUENCE CHARACTERISTICS:
LENGTH: 26 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION: SEQ ID N0: 12 gagccgatag acaaggaact gtatcc 26 INFORMATION FOR SEQ ID NO: 13 SEQUENCE CHARACTERISTICS:
LENGTH: 26 TYPE: DNA
' CA 02317789 2001-02-21 ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION: SEQ ID N0: 13 gagccgatag acaaggaagt gtatcc 26 INFORMATION FOR SEQ ID N0: 14 SEQUENCE CHARACTERISTICS:
LENGTH: 26 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION: SEQ ID N0: 14 gagccgatag acaaggaaat gtatcc 26 INFORMATION FOR SEQ ID N0: 15 SEQUENCE CHARACTERISTICS:
LENGTH: 26 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION FOR ID N0: 15 gagccgatag acaagggact gtatcc 26 INFORMATION FOR SEQ ID N0: 16 SEQUENCE CHARACTERISTICS:
LENGTH:26 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION: SEQ ID N0: 16 gagccgatag acaaggacct gtatcc 26 INFORMATION FOR SEQ ID NO: 17 SEQUENCE CHARACTERISTICS:
LENGTH: 26 TYPE: DNA
ORIGNAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION: SEQ ID N0: 17 gagccgatag acaagggcct gtatcc 26 INFORMATION FOR SEQ ID NO: 18 SEQUENCE CHARACTERISTICS:
LENGTH: 26 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION: SEQ ID NO: 18 gagccgatag acaaggagct gtatcc 26 INFORMATION FOR SEQ ID N0: 19 SEQUENCE CHARACTERISTICS:
LENGTH: 26 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: sequencing primer for protease gene SEQUENCE DESCRIPTION: SEQ ID N0: 19 gagccgatag acaaggggct gtatcc 26 INFORMATION FOR SEQ ID N0: 20 SEQUENCE CHARACTERISTICS:
LENGTH: 26 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: RT1 forward primer SEQUENCE DESCRIPTION:SEQ ID NO: 20 gttaaacaat ggccattgac agaaga 26 INFORMATION FOR SEQ ID N0: 21 SEQUENCE CHARACTERISTICS:
LENGTH: 24 TYPE: DNA
ORIGNAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: RT1 reverse primer SEQUENCE DESCRIPTION:SEQ ID N0: 21 ggaatattgc tggtgatcct ttcc 24 INFORMATION FOR SEQ ID N0: 22 SEQUENCE CHARACTERISTICS:
LENGTH: 22 TYPE: DNA
ORGANISM: Human immunodeficiency virus OTHER INFORMATION: RT1 forward primer SEQUENCE DESCRIPTION:SEQ ID N0: 22 gttaaacaat ggccattgac ag 22 INFORMATION FOR SEQ ID N0: 23 SEQUENCE CHARACTERISTICS:
LENGTH: 27 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: RT2 forward sequencing primer SEQUENCE DESCRIPTION: SEQ ID NO: 23 gaagtatact gcatttacca tacctag 27 INFORMATION FOR SEQ ID N0: 24 SEQUENCE CHARACTERISTICS:
LENGTH: 27 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: RT2 forward pimer SEQUENCE DESCRIPTION: SEQ ID N0: 24 gaagtatact gcatttacta tacctag 27 INFORMATION FOR SEQ ID N0: 25 SEQUENCE CHARACTERISTICS:
LENGTH: 27 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: RT2 forward primer SEQUENCE DESCRIPTION: SEQ ID N0: 25 aaagtatact gcattcacca tacctag 27 INFORMATION FOR SEQ ID N0: 26 SEQUENCE CHARACTERSTICS:
LENGTH: 27 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: RT2 forward primer SEQUENCE DESCRIPTION: SEQ ID N0: 26 gaaatatacc gcatttacca tayctag 27 INFORMATION FOR SEQ ID N0: 27 SEQUENCE CHARACTERISTICS:
LENGTH: 24 TYPE: DNA
ORGANISM: Human immunodeficiency virus OTHER INFORMATION: RT2 reverse primer SEQUENCE DESCRIPTION: SEQ ID N0: 27 tctgtatgtc attgacagtc cagc 24 INFORMATION FOR SEQ ID N0: 28 SEQUENCE CHARACTERISTICS:
LENGTH: 24 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: RT2 reverse primer SEQUENCE DESCRIPTION: SEQ ID N0: 28 tctgtatatc attgacagtc cagt 24 INFORMATION FOR SEQ ID N0: 29 SEQUENCE CHARACTERISTICS:
LENGTH: 24 TYPE: DNA
ORIGNAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: RT2 reverse primer SEQUENCE DESCRIPTION:SEQ ID N0: 29 tctgtatatc attgacagtc cagc 24 INFORMATION FOR SEQ ID N0: 30 SEQUENCE CHARACTERISTICS:
LENGTH: 25 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: RT2 reverse primer SEQUENCE DESCRIPTION: SEQ ID NO: 30 ttctgtatgt cattgacagt ccagc ~ 25 INFORMATION SEQ ID N0: 31 SEQUENCE CHARACTERISTICS:
LENGTH: 21 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: P2 forward primer SEQUENCE DESCRIPTION: SEQ ID N0: 31 ttccctcaga tcactctttg g 21 INFORMATION FOR SEQ ID N0: 32 SEQUENCE CHARACTERISTICS:
LENGTH:21 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMTAION: P2 forward primer SEQUENCE DESCRIPTION: SEQ ID N0: 32 ttccctcaaa tcactctttg g 21 INFORMATION FOR SEQ ID N0: 33 SEQUENCE CHARACTERISTICS:
LENGTH: 21 TYPE: DNA
ORIGINAL SOURCE:
ORGANISM: Human immunodeficiency virus FEATURE:
OTHER INFORMATION: P2 reverse primer SEQUENCE DESCRIPTION: SEQ ID N0: 33 acttttgggc catccattcc t 21
Claims (5)
1. A method for determining the genetic type of HIV-1 present in a sample containing HIV-1, comprising determining the positions of the nucleotides within the protease and reverse transcriptase genes and comparing these positions to the positions of nucleotides in known genetic types, using at least one primer selected from the group consisting of SEQ ID Nos. 6, 7, 23, 24, 25, 26, 31, 32 and 33.
2. The method of claim 1, wherein the positions of the nucleotides are determined by performing a cycled reaction that generates both forward and reverse sequencing fragments using two primers, each primer labeled with a different and distinguishable detectable label.
3. The method of claim 2, wherein the label is a fluorescent label.
4. A kit for performing sequencing on an HIV-1 genome, comprising a plurality of termination mixtures, each of said termination mixtures including one of a plurality of primer pairs, each pair flanking a different region of the HIV-1 genome, the pairs together flanking substantially all of the protease and reverse transcriptase genes, and at least one member of each pair being labeled with a detectable label, wherein the primers are selected from the group consisting of:
(a) a primer pair for sequencing a portion of the protease gene comprising a forward primer selected from the group consisting of SEQ ID Nos. 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19, and a reverse primer having the sequence of SEQ ID
No. 8;
(b) a primer pair for sequencing a portion of the protease gene comprising a forward primer selected from the group consisting of SEQ ID Nos.31 and 32, and a reverse primer having the sequence of SEQ ID no. 33;
(c) a primer pair for sequencing a portion of the reverse transcriptase gene comprising a forward primer selected from the group consisting of SEQ ID Nos.
23, 24, 25 and 26, and a reverse primer selected from the group consisting of SEQ ID Nos.
27, 28, 29 and 30; and (d) combinations of the primer pairs set forth in paragraphs (a) - (c).
(a) a primer pair for sequencing a portion of the protease gene comprising a forward primer selected from the group consisting of SEQ ID Nos. 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19, and a reverse primer having the sequence of SEQ ID
No. 8;
(b) a primer pair for sequencing a portion of the protease gene comprising a forward primer selected from the group consisting of SEQ ID Nos.31 and 32, and a reverse primer having the sequence of SEQ ID no. 33;
(c) a primer pair for sequencing a portion of the reverse transcriptase gene comprising a forward primer selected from the group consisting of SEQ ID Nos.
23, 24, 25 and 26, and a reverse primer selected from the group consisting of SEQ ID Nos.
27, 28, 29 and 30; and (d) combinations of the primer pairs set forth in paragraphs (a) - (c).
5. The kit according to claim 4, wherein the primers in each primer pair are labeled with different and spectroscopically distinguishable fluorescent labels.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US09/418,720 US6265152B1 (en) | 1995-12-22 | 1999-10-15 | Method and kit for evaluation of HIV mutations |
| US09/418,720 | 1999-10-15 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2317789A1 true CA2317789A1 (en) | 2001-04-15 |
Family
ID=23659312
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002317789A Abandoned CA2317789A1 (en) | 1999-10-15 | 2000-09-14 | Method and kit for evaluation of hiv mutations |
Country Status (5)
| Country | Link |
|---|---|
| US (2) | US6265152B1 (en) |
| AU (1) | AU5793500A (en) |
| CA (1) | CA2317789A1 (en) |
| FR (1) | FR2800094B1 (en) |
| GB (1) | GB2356703B (en) |
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|---|---|---|---|---|
| US6265152B1 (en) * | 1995-12-22 | 2001-07-24 | Visible Genetics Inc. | Method and kit for evaluation of HIV mutations |
| US6379957B1 (en) * | 1998-09-21 | 2002-04-30 | Leslie A. Johnston-Dow | Methods for HIV sequencing and genotyping |
| ATE456677T1 (en) * | 1999-07-09 | 2010-02-15 | Gen Probe Inc | HIV-1 DETECTION USING NUCLEIC ACID AMPLIFICATION |
| US6582920B2 (en) | 2000-09-01 | 2003-06-24 | Gen-Probe Incorporated | Amplification of HIV-1 RT sequences for detection of sequences associated with drug-resistance mutations |
| US20040157238A1 (en) * | 2002-09-20 | 2004-08-12 | Quinn John J. | Method for detection of multiple nucleic acid sequence variations |
| WO2005019424A2 (en) * | 2003-08-18 | 2005-03-03 | Wright State University | Retroviral vector and cell-based assay for measuring the mutation rate of retroviruses employing same |
| US20160258010A1 (en) * | 2013-10-18 | 2016-09-08 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Allele specific pcr assay for detection of nucleotide variants |
| EP3063272A2 (en) | 2013-10-30 | 2016-09-07 | Green Life Biotech, LLC | Pathogenesis quantification systems and treatment methods for citrus greening blight |
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| CA2159044A1 (en) * | 1994-09-26 | 1996-03-27 | Falko-Guenter Falkner | Method of quantitating nucleic acids |
| US5888736A (en) * | 1995-12-22 | 1999-03-30 | Visible Genetics, Inc. | Method, compositions and kit for detection and identification of microorganisms |
| US5795722A (en) | 1997-03-18 | 1998-08-18 | Visible Genetics Inc. | Method and kit for quantitation and nucleic acid sequencing of nucleic acid analytes in a sample |
| US6265152B1 (en) * | 1995-12-22 | 2001-07-24 | Visible Genetics Inc. | Method and kit for evaluation of HIV mutations |
| WO1997027332A1 (en) * | 1996-01-26 | 1997-07-31 | Innogenetics N.V. | Method for detection of drug-induced mutations in the reverse transcriptase gene |
| US5837464A (en) * | 1996-01-29 | 1998-11-17 | Virologic, Inc. | Compositions and methods for determining anti-viral drug susceptibility and resistance and anti-viral drug screening |
| US6114312A (en) * | 1997-03-07 | 2000-09-05 | Research Institute For Genetic And Human Therapy (R.I.G.H.T.) | Method of inhibiting human immunodeficiency virus by combined use of hydroxyurea, a nucleoside analog, and a protease inhibitor |
| WO1999016910A1 (en) * | 1997-09-26 | 1999-04-08 | Visible Genetics Inc. | Method and kit for evaluation of hiv mutations |
| WO1999067428A2 (en) * | 1998-06-24 | 1999-12-29 | Innogenetics N.V. | Method for detection of drug-selected mutations in the hiv protease gene |
-
1999
- 1999-10-15 US US09/418,720 patent/US6265152B1/en not_active Expired - Lifetime
-
2000
- 2000-09-13 AU AU57935/00A patent/AU5793500A/en not_active Abandoned
- 2000-09-14 CA CA002317789A patent/CA2317789A1/en not_active Abandoned
- 2000-09-20 GB GB0023049A patent/GB2356703B/en not_active Expired - Fee Related
- 2000-10-11 FR FR0012980A patent/FR2800094B1/en not_active Expired - Fee Related
-
2001
- 2001-03-27 US US09/818,182 patent/US6653107B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| US20030165815A1 (en) | 2003-09-04 |
| GB0023049D0 (en) | 2000-11-01 |
| FR2800094B1 (en) | 2004-11-26 |
| FR2800094A1 (en) | 2001-04-27 |
| AU5793500A (en) | 2001-04-26 |
| GB2356703B (en) | 2004-07-21 |
| GB2356703A (en) | 2001-05-30 |
| US6653107B2 (en) | 2003-11-25 |
| US6265152B1 (en) | 2001-07-24 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |