GB2395787A - Method and kit for evaluation of HIV mutations - Google Patents
Method and kit for evaluation of HIV mutations Download PDFInfo
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- GB2395787A GB2395787A GB0402068A GB0402068A GB2395787A GB 2395787 A GB2395787 A GB 2395787A GB 0402068 A GB0402068 A GB 0402068A GB 0402068 A GB0402068 A GB 0402068A GB 2395787 A GB2395787 A GB 2395787A
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Abstract
A method for determining the genetic type of HIV-1 present in a sample containing HIV-1, comprising sequencing at least part of the reverse transcriptase gene utilising at least one of a number of specific primers. A kit for performing sequencing on an HIV-1 genome is also provided, comprising a plurality of sequence specific primer pairs. In a preferred embodiment, the primers used in the method and kit are labelled with a fluorescent label.
Description
GB 2395787 A continuation (74) Agent and/or Address for Service: (58)
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Urquhart-Dykes & Lord INT CL7 C12Q Alexandra House, 1 Alexandra Road, Other: GAS-ONLINE, DGENE, BIOSIS, EMBASE, SWANSEA, SA1 BED, United Kingdom MEDLINE, SCISEARCH, EPODOC, WPI, PAJ
to -1 METHOD AND KIT FOR F.VALIJATION OF HIV MU1 ATIONS
BACKGROUND OF TH F. 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 5 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 10 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 15 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 eleetrophoretically separated on a non-denaturing eleetrophoresis gel. This pattern depends on a combination of the size of the fragments and of the three-dimensional 20 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 immunodeficiency virus (HTV).
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 HlV-infected sample. A test is performed in which the sequence is determined in the 3'-direction for all 30 tour 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 tour 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'dircction, all in the 5'-direction, or as a 5 combination of the two strands.; BRIEF DESCRIPTION OF THE DRAWINGS
Fig. I shows a schematic representation of the invention.
10 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.
I. "Allele" refers to a specific version of a nucleotide sequence at a polymorphic genetic locus. 15 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 20 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.
25 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 allelespecific ligation assays.
30 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
-3- generally in International latent Publication No. WO 97/2365O, certain alleles of a genetic locus may be distinguishable on the basis of idcntilication of the location of less than lLur, and often only one nucleotide. This finding allows the development of the present method for improved allele identification within the highly polyTIlorphic HTV genome.
5 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 clectrophoresis. (see Chp 7.6, Current Protocols in 10 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 15 bases of this specific type. The information provided by the four lanes is therefore combined in known sequencing procedures to arrive 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: 20 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 t1IV- 1 (the primer regions are not included in this range). 2) Sequencing reactions are perfonned. 3) Finally, the sequencing ladders are analyzed, preferably using the OpenGenc_ System: the MicroGene Clipper_ or Long-Read 25 TowerrM DNA Sequencers, GeneObjects_ and GeneLibrarian_ Software.
Fig. I shows one embodiment of the method of the invention schematically.
As shown, an RNA sample is obtained and treated by reverse transcriptasePCR (RT PCR) to produce an amplicon of approximately 1.3 kbase pairs spanning the proteasc and reverse transcriptase genes of the HIV genome from a target cell. This reaction can be 30 performed using, for example, the TITAN_ One-Tube RT-PCR system from Boehringer Mannhcim (Cat. No. 1 855 476 or l 882 382) using the following primers: forward primer set:
-4 AAGCAGGAGC CGATAGACAA GGSEQ ID No. 1 AAGCAGGAGC HGAWAGACAR GGSEQ ID No. 2 CAGCAGGAAC CGAGGGACAA GGSEQ ID No. 3 reverse primer set: 5 CTAYTARGTC TTTTGWTGGG TCATA-EQ ID No. 4 GCTATTAAGT CTTTTGATGG GTCASEQ 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 MgCI2 at 25 "C) and a polymerase enzyme I O such as Taq FS (Perkin Elmcr/Applied Biosystems (:at No. 402070) I his polymerase has a high rate of incorporartion of dideoxynucleotidc 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 1 5 primers: forward primers: GAGCCRATAG ACAAGGAAYT RTAT SEQ ID No. 6 GAGMCGATAG ACAAGGRVCT GTAT SEQ ID No. 7 reverse primers: 20 ACTTTTGGGC CATCCATTCC T SEQ ID No. 8 Other forward primers which could be used at this step include: GAGCCGATAG ACAAGGAACT ATATCC SEQ ID No. 9 GAGCCGATAG ACAAGGAAGT ATATCC SEQ ID No. 10 25 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 30 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 arc used as follows: RT1 Primers forward: GTTAAACAAT GGCCATTGAC AGAAGA SEQ ID No. 20 5 reverse:; GGAATATTGC TGGTGATCCT TTCC SEQ ID No. 21 alternate forward: GTTAAACAAT GGCCATTGAC AG SEQ ID No. 22 10 RT2 Primers forward: GAAGTATACT GCATTTACCA TACCTAG SEQ ID No. 23 GAAGTATACT GCATTTACTA TACCTAG SEQ ID No. 24 AAAGTATACT GCATTCACCA TACCTAG SEQ ID No. 25 15 GAAATATACC GCATTTACCA TAYCTAG SEQ ID No. 26 reverse: TCTGTATGTC ATTGACAGTC CAGC SEQ ID No. 27 alternate reverse: TCTGTATATC ATTGACAGTC CAGT SEQ ID No. 28 20 TCTGTATATC ATTGACAGTC CAGC SEQ ID No. 29 TTCTGTATGT CATTGACAGT CCAGC SEQ ID No. 30 P2 Primers forward: 25 TTCCCTCAGA TCACTCTTTG G SEQ ID No. 31 TTCCCTCAAA TCACTCTTTG G SEQ ID No. 32 reverse: ACTTTTGGGC CATCCATTCC T SEQ ID No. 33 30 The P2 forward primers arc 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 Genctics Inc. MicroGene Clipper or Long-Read Tower
-6 scquencers), both the forward and reverse primers arc 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 5 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 *agments can be produced in a single reaction using distinctively labeled forward and reverse primers, or in 10 separate reactions depending on the nature of the detection system being cmploycd.
Reagents suitable for practicing the method of the invention are suitably packaged in kit fonm. Thus, the invention provides a kit for analyzing the genetic type of an HIV- I gene in a sample comprising: a kit for pertonming four base sequencing on HIV I comprising a plurality of A, C, G and T terminations mixtures, each of said termination 15 mixtures including one of a plurality of primer pairs, each pair flanking a different region of the HIV- I 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 suLkits for pertonming four base sequencing may be included when intermediate and final assays on one strand and both strands are desired.
20 As used herein, the tenm "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 suLkit for performing A and T sequencing on HIV-1 may also be provided 25 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 perfonning 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 30 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.
-7 EXAMPLE 1
The RT-PCR is done on the HIV-1 RNA using a blend of enzymes forming RTPCR Master Mixes described below to conduct six RT-PCT reactions. This RTPCR 5 is done on the RNA preparation obtained using the QlAmp Viral RNA kit from Qiagen. It can also be done on the RNA extract for the NucliSense_ (formerly known as NASBA) HIV Viral Load from Organon Teknica.
All the reagents, tubes, tips, and other material needs to be RNase-free.
10 The recipe is made t'or 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-t'ree, and prepare the 15 RT-PCR Master Mix I (enough for 6 tubes, i.c. 6 samples) by adding the following ingredient in the order listed: RT-PCR MASTER MIX I
7 al of 80 mM DTT 10.5 p1 of RNase-free dNTP at 10 mM each damp 20 21 Ill of forward PCR primer at 28 rum.
21 Ill of reverse PCR primer at 28 I1M 3.5 Ill of Rnase-inhibitor from Roche Molecular Biochemicals, catalog # 799 025 (10,000 U) 25 Take a 0.2 ml sterile, RNase-free, centrifuge tube, RNase-t'ree, and prepare the RTPCR Master Mix II (enough for 6 tubes) by adding the following ingredient in the order listed: RT-PCR MASTER MIX II
70 al of 4x RT-PCR Buffer (280 mM Tris Hcl, 9.2 mM MgCI2, 60 mM (Nl14) 2SO4, 100 30 1lg/ml Acetylated BSA from Lite Tech, CA, pH 8.60 at 25"C) 3. 5 Ill of RNase Inhibitor at40 U/ 7 pi of Superscript II
-8 8.75 Ill of Expand High Fidelity Enzyme System Enzyme Mix from Roche Moleeular Biochemicals, catalog # 1 7333 818 8.75 Ill of AmpliTaq from Roche Molecular Systems.
Take one strip of 6 thin wall tubes. Add 9 al of MASTER MIX I in each tube.
5 Add 17 Ill 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 Ill 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 10 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 15 3= 50.0 for 1:00:00 4= 94.0 for2:00 5= 1.0 /s to 94.0 6= 94.0 for 0:30 7= 1.0 /s to 57.0 20 8= 57.0 for 0:30 9= 1.0 /s to 68.0 10= 68.0 for 2:00 I l=Goto 5, 19 times 1 2= 1.0 /s to 94.0 25 13= 94.0 for 0:30 14= 1.0/s to 60.0 15= 60.0 for 0:30 16=1.0'/sto 68.0 17= 68.0 for2:30 30 1 8=Goto 12, 16 times 19=68.0 for 7:00 20= 4.0 for ever
21 =End EXAMPLE 2
To determine the sequence of amplicon, 7 al of each terminator mix (16 when 5 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 Ill of butter (260 mM Tris-HCI, pH 8.3 at 25 C, 32.5 mM MgCI2) 475 Ill of sterile water 22.5 pi enzyme blend of AmpliTa4 FS from Roche Molecular Systems 15 U/lll and 27 I O U/,ul pyrophosphotase 5 Ill 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 15 1= 94.0 for 5:00 2= 1.OU/s to 94.0 3= 94.0 for 0:20 4= 1.0 /s to 56.0 5= 56.0 for 0:20 20 6= 1.0 /sto 70.0 7= 70. 0 for 1:30 8=Goto 2, 29 times 9= 70.0' for 5:00 10= 4.0 for ever 25 11=End I'ermination mixes for two dye systems Protease A-Mix: 1.07,uM ddATP; 643 I1M dATP; 643,uM dCTP; 643,uM dGTP; 643 I1M dTTP; 30 330 nM total of forward primers and 330 nM total of reverse primers; I mM TrisHCI, pH 8.0 at 25 C, 0. I mM EDTA.
C-Mix: 2.14,uM ddCTP; 643 I1M dATP; 643 EM dCTP; 643 I1M dGTP; 643 I1M dTTP;
-lo- - - 330 nM total of forward primers and 330 nM total of reverse primers; I mM Tris-HCl, pH 8.0 at 25"C, 0.1 mM EDTA.
G-Mix: 2.14 I1M ddG'I'P; 643 I1M dATP; 643 I1M dCTP; 643 I1M dGTP; 643 I1M dTTP; 330 nM total of t'orward primers and 330 nM total of reverse pnmers; I mM Tris-HCI, pH 5 8.0 at 25"C, 0.1 mM EDTA.; T-Mix: 2.14 I1M ddTTP; 643 I1M dATP; 643 M dCTP; 643 I1M dGTP; 643 I1M dTTP; 330 nM total of forward primers and 330 nM total of reverse primers; 1 mM Tris-HCl, pH 8.0 at 25 C, 0.1 mM EDTA.
Both primers are labeled, for example with CyS.O and CyS.5, respectively.
First RT region A-Mix: 1.07 I1M ddATP; 643 I1M dATP; 643 I1M dCTP; 643 M dGTP; 643 I1M d'l'TP; 330 nM total of forward primers and 330 nM total of reverse primers; 1 mM Tris-HCl, pH 8.0 at 25 C, 0.1 mM EDTA.
15 C-Mix: 2.14 I1M ddCTP; 643 I1M dATP; 643 M dCTP; 643,uM dGTP; 643,uM dTTP; 330 nM total of forward primers and 330 nM total of reverse primers; I mM Tris-HCI, pH 8.0 at 25 C, 0.1 mM EDTA.
G-Mix: 2.14 I1M ddGTP; 643 I1M dATP; 643,uM dCTP; 643 M dGTP; 643 I1M dTTP; 330 nM total of forward primers and 330 nM total of reverse primers; 1 mM Tris-HCl, pH 20 8.0 at 25 C, 0.1 mM EDTA.
T-Mix: 2.14 uM ddTTP; 643 I1M dATP; 643 I1M dCTP; 643,uM dGTP; 643 I1M 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.
25 Second reverse transcriptasc region A-Mix: 1.07,uM ddATP; 643 I1M dATP; 643 I1M dCTP; 643,uM dGTP; 643 I1M dTTP; 330 nM total of forward primers and 330 nM total of reverse primers C-Mix: 2.14,uM ddCTP; 643 I1M dATP; 643 I1M dCTP; 643 M dGTP; 643 M dTTP; 330 nM total of forward primers and 330 nM total of reverse primers 30 G-Mix: 2.14 I1M ddGTP; 643 M dATP; 643 I1M dCTP; 643 I1M dGTP; 643 I1M dTTP; 330 nM total of forward primers and 330 nM total of reverse primers T-Mix: 2.14 I1M ddTTP; 643 I1M dATP; 643, uM dCTP; 643,uM dGTP; 643 M dTTP;
-1 1 330 nM total of forward primers and 330 nM total of reverse primers Both primers are labeled, for example with CyS.O and Cy5.5, respectively.
P2 protease region 5 A-Mix: 1.07 M ddATP; 643 I1M dATP; 643 I1M dC'l'P; 643 I1M dGTP, 643 M dTTP; 330 nM total of forward primers and 330 nM total of reverse primers C-Mix: 2.14 I1M ddCTP; 643 I1M dATP; 643 I1M dCTP; 643 M dGTP; 643 I1M dTTP; 330 nM total of forward primers and 330 nM total of reverse primers G-Mix: 2.14 I1M ddGTP; 643 I1M dATP; 643 I1M dCTP; 643 I1M dGTP; 643 M dTTP; 10 330 nM total of f'orward primers and 330 nM total of reverse primers T-Mix: 2.14,uM ddTTP; 643 I1M dATP; 643, uM dCTP; 643 I1M 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.
!a SEQUENCE LISTING
- <110> Visible Genetics Inc <120> Method And Kit For Evaluation Of HIV Mutations <130> P450287
<140> <141> <150> US 09/418720
<151> 1999-10-15
<160> 33
<170> PatentIn Ver. 2.1 <210> 1
<211> 22
<212> DNA
<213> Human immunodeficiency virus <400> 1
aagcaggagc cgatagacaa gg 22 <210> 2
<211> 22
<212> DNA
<213> Human immunodeficiency virus <400> 2
aagcaggagc hgawagacar gg 22 <210> 3
<211> 22
<212> DNA
<213> Human immunodeficiency virus <400> 3
cagcaggaac cgagggacaa gg 22 <210> 4
<211> 25
<212> DNA
<213> Human immunodeficiency virus <400> 4
ctaytargtc ttttgwtggg tcata 25 <210> 5
<211> 24
<212> DNA
<213> Human immunodeficiency virus <400> 5
gctattaagt cttttgaLgg gtca 24 <210> 6
<211> 24
<212> DNA
<213> Human immunodeficiency virus <400> 6
gagacratag acaaggaayt rtat 24 <210> 7
<211> 24
<212> DNA
c213> Human immunodeficiency virus <400> 7
gagmcgatag acaaggrvct Stat 24 <210> 8
<211> 21
<212> DNA
<213> Human immunodeficiency virus <400> 8
acttttgggc catccattcc t 21 <210> 9
<211> 26
<212> DNA
<213> Human immunodeficiency virus <400> 9
gagacgatag acaaggaact atatcc 26 <210> 10
<211> 26
<212> DNA
<213> Human immunodeficiency virus <400> 10
gagccgatag acaaggaagt atatcc26 _ <210> 11
<211> 26
<212> DNA
<213> Human immunodeficiency virus <400> 11
gagccgatag acaaggaaat atatcc 26 <210> 12
<211> 26
<212> DNA
<213> Human immunodeficiency virus <400> 12
gagccgatag acaaggaact gtatcc26 <210> 13
<211> 26
<212> DNA
<213> Human immunodeficiency virus <400> 13
gagacgatag acaaggaagt gtatcc26 <210> 14
<211> 26
<212> DNA
<213> Human immunodeficiency virus <400> 14
gagacgatag acaaggaaat gtatcc26 <210> 15
<211> 26
<212> DNA
<213> Human immunodeficiency virus <400> 15
gagccgatag acaagggact gLatcc26 <210> 16
<211> 26
<212> DNA
<213> Human immunodeficiency virus <400> 16
IS gagccgatag acaaggacct gtatcc 26 <210> 17
<211> 26
<212> DNA
<213> Human immunodeficiency virus <400> 17
gagccgatag acaagggcct gtatcc 26 <210> 18
<211> 26
<212> DNA
<213> Human immunodeficiency virus <400> 18
gagccgatag acaaggagct gtatcc 26 <210> 19
<211> 26
<212> DNA
<213> Human immunodeficiency virus c400> 19 gagccgatag acaaggggct gtatcc 26 <210> 20
<211> 26
<212> DNA
<213> Human immunodeficiency virus <400> 20
ghtaaacaat ggccattgac agaaga 26 <210> 21
<211> 24
<212> DNA
<213> Human immunodeficiency virus <400> 21
ggaatattgc tggtgatcct tCcc 24 <210> 22
<211> 22
<212> DNA
<213> Human immunodeficiency virus
<a O> 22 gttaaacaat ggccattgac ag 22 <210> 23
<211> 27
<212> DNA
<213> Human immunodeficiency virus <400> 23
gaagtatact gcatttacca tacctag 27 <210> 24
<211> 27
<212> DNA
<213> Human immunodeficiency virus <400> 24
gaagtatact gcathtacta tacctag 27 <210> 25
<211> 27
<212> DNA
<213> Human immunodeficiency virus <400> 25
aaagLatact gcattcacca tacctag 27 <210> 26
<211> 27
<212> DNA
<213> Human immunodeficiency virus <400> 26
gaaatatacc gcatttacca tayctag 27 <210> 27
<211> 24
<212> DNA
<213> Human immunodeficiency virus <400> 27
tctgtaLgtc attgacagec cage 24 <210> 28
<211> 24
<212> DNA
<213> Human immunodeficiency virus
<400> 28
tctgtatatc attgacagtc cagt 24 <210> 29
<211> 24
<212> DNA
<213> Human immunodeficiency virus <400> 29
tatgtatatc attgacagtc cage 24 <210> 30
<211> 25
<212> DNA
<213> Human immunodeficiency virus <400> 30
tCctgtatgt cattgacagt ccagc 25 <210> 31
<211> 21
<212> DNA
<213> Human immunodeficiency virus <400> 31
ttccctcaga tcactctttg g 21 <210> 32
<211> 21
<212> DNA
<213> Human immunodeficiency virus <400> 32
ttccctcaaa tcactchttg g 21 <210> 33
<211> 21
<212> DNA
<213> Human immunodeficiency virus <400> 33
ackthtgggc catccattcc t 21
Claims (5)
1. A method for determining the genetic type of HIV-1 present in a sample containing HIV-I, comprising determining the positions of the nucleotides within the reverse 5 transcriptase gene 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 No's 27, 28, 29 and 30.
2. The method of claim 1, wherein the positions of the nucleotides are determined by lo performing a cycled reaction that generates both forward and reverse sequencing fragments using two primers, each primer labelled 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 H[V-1 genome, comprising a plurality of termination mixtures, each of said termination mixtures including one of a plurality of primerpairs, each pair flanking a differentregion ofthe HIV-1 genome, and at least one member of each pair being labelled with a detectable label, wherein the primers are 20 selected from the group consisting of: a primer pair for sequencing a portion of the reverse transcnptase gene comprising a forward primer selected from the group consisting of SEQ ID No's 23, 24, 25 and 26, and a reverse primer selected from the group consisting of SEQ [D No's 27, 28, 29 and 30; or 2s combinations of the primer pairs.
5. The kit according to claim 4, wherein the primers in each primer pair are labelled 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 |
| GB0201236A GB2367616B (en) | 1999-06-23 | 2000-06-22 | Water detection and source identification methods for structures using electromagnetic radiation spectroscopy |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| GB0402068D0 GB0402068D0 (en) | 2004-03-03 |
| GB2395787A true GB2395787A (en) | 2004-06-02 |
Family
ID=32232383
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB0402068A Withdrawn GB2395787A (en) | 1999-10-15 | 2000-09-20 | Method and kit for evaluation of HIV mutations |
Country Status (1)
| Country | Link |
|---|---|
| GB (1) | GB2395787A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999016910A1 (en) * | 1997-09-26 | 1999-04-08 | Visible Genetics Inc. | Method and kit for evaluation of hiv mutations |
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2000
- 2000-09-20 GB GB0402068A patent/GB2395787A/en not_active Withdrawn
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999016910A1 (en) * | 1997-09-26 | 1999-04-08 | Visible Genetics Inc. | Method and kit for evaluation of hiv mutations |
Non-Patent Citations (5)
| Title |
|---|
| AIDS Research & Human Retroviruses (1998), Vol 14(10), pp 869-876, "Comparative performance of high-density...", Gunthard et al * |
| AIDS Research & Human Retroviruses (Sep. 1999), Vol 15(14), pP 1287-1292, "Rapid detection of the HIV Type 1...", Ross et al * |
| Electrophoresis (1992); Vol 13(8); pp 547-551, "Automated magnetic preparation of DNA...", Wahlberg et al * |
| Journal of Clinical Microbiology (1995), Vol 33(2), pp 342-347, "Specific, sensitive and rapid assay...", Frenkel et al * |
| Virology (1996) Vol 219(2), pp 407-416, "Natural variation in HIV-1 protease,...", Barrie et al * |
Also Published As
| Publication number | Publication date |
|---|---|
| GB0402068D0 (en) | 2004-03-03 |
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| 732E | Amendments to the register in respect of changes of name or changes affecting rights (sect. 32/1977) | ||
| WAP | Application withdrawn, taken to be withdrawn or refused ** after publication under section 16(1) |