GB2395007A - Method and kit for evaluation of HIV mutations - Google Patents

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 protease 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

METHOD ANn KIT FOR EVALUA'I'ION ()F HIV MIJ'I'A'I'IONS BAC'K(>,ROUNI) OF

THI. INVENTIC)N

Genetic testing to determine the presence ot'or a susceptibility to a disease condition ot't'crs incredible opportunities for improved medical care, and.the potential for 5 such testing increases almost daily as ever increasing numbers ot'diseasc-associatcd genes and/or mutations are identified. A major hurdle which must be overcome to realize this potential, however, is the high cost ot'testing. This is particularly true in the case ot' highly polyTnophic genes where the need to test t'or a large number of variations may make the test procedure appear to be so expensive that routine testing can never be 1 0 achieved.

Testing for changes in DNA sequcoce 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 confonnational polymorphism" ( "SSCP") described by 15 Orita et al., Genomics 5: 874-879 ( 1989), or by a modification thereof ret'errcd to a didcoxy-fingerprinting ("ddF") described by Sarkar et al., Genomic.s 13: 4410443 (1992).

SSCP and ddF both evaluate the pattern of bands created when DNA fragments are electrophorctically separated on a non-denaturing electrophoresis gel. This pattern depends on a combination of the siz.c of the t'ragmcnts 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 (HIV).

SUMMARY OF THE INVI:NTIC)N

I'he method ot'the invention provides a method t'or obtaining information about the allelic type of a sample of genetic material derived t'rom an HlV-infected sample. A test is perf'onned in which the sequence is determined in the 3'-direction t'or 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 four bases.

To pcrtonn the mcthotl of the invcution, reagents sitahlc for the tests are suitably packaged as a kit. I he l;it contains rcagcnt.s for pcrlonning a tour-base sequence dctennination on one or both strands ot the target 1)NA. One-stranded sequence I determination could be performed all in the 3'-dircction, all in the 5'-dh-cction, or as a I 5 combination of the two strands.

BRIEF L)ES(:RIP I ION Ol THE DRAWINGS Fig. I shows a schematic representation of the invention.

1 () DE I AILED DESC'RIP I ION OF TILE INVENTION

While the terminology used in this application is standard within the art, the following definitions of certain terms are provided to assure clarity.

1. "Allele" refers to a specific version ot 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. Dideoxynuclcotides 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 sutticient 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 nuclcotides of a polymorphic genetic locus in order to determine which allele is present in a specific patient sample. As disclosed

encrally in International Patent Publication No. W0')7/9,(5(), certain alleles,t'a genetic locus may be distinguishable on the basis ol'idcntitication of the location ol'lcss than tour, and ot'ten only one nucleotitle. This finding tallows the cieelopment ot'thc present method for improved allele identification within the highly polymorphic HIV genome.

5 Traditionally, it'sequencing were going, to be used to evaluate the allelie type ot'a polymorphic gene, four dideoxy nucleotide "sequencing" reactions ot'the type described by Sanger et al. (I'roc. Natl. Acad. Sci. USA 74: 5463-5467 (1377)) would be run on the sample concurrently, and the products ot'the tour reactions would then be analyzed by polyacrylamide gel clectrophoresis. (see Chp 7.6, Current Protocols in 1() Molecular Biology, Eds. Ausubel, I:.M. et all (John Wiley 8; Sons; 19)5)) In this \vell known technique, each of the t'our sequencing reactions generates a plurality ot'primer extension products, all of which end with a specific type of didcoxynucleotide. Each lane on the electrophoresis gel thus reflects the positions of one type ot'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 ot'a good portion of the diagnostically relevant protease and reverse transcriptase genes is obtained in three steps: ?() I) cDNA is generated from the RNA present in the sample, and amplified, preferably across a region extending t'rom 6 codons before the protease up to codon 335 ot'the reverse transcriptase of HIV-I (the primer regions are not included in this range). 2) Sequencing reactions are performed. 3) Finally, the sequencing ladders are analyzed, preferably using the OpenGene_ System: the MicroGene Clipper_ or Long-Read 95 Towered DNA Sequencers, GeneObjects_ and Genel. ibrarian_ Software.

Fig. I shows one embodiment ot'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 protease and reverse transcriptase francs of the H IV gcnome t'rom a target cell. This reaction can be 30 perf'onned using, t'or example, the TITAN_ One-Tube RT-PCR system Tom Boehringcr Mannheim (Cat. No. 1 855 476 or 1 882 382) using the tallowing 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 but'fer (260 mM Tris-HCL, pH ú.3; 32.5 mM MgCI? at 25 "C) and a polymerase enzyme I () sueh as Taq FS (Perkin F:lmer/Applied lliosystems C:at No. 4()()7()) This polymerase has a high rate of incorporartion of dideoxynucleotide relateive to the incorporation rate ot; tor example, conventional 1'aq polymerase. 'I'his 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 t'orward 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

( I or thc rcversc transcriptase gcnc, thrcc sets ot'primers are used as fUllows: R'l'l Primers forward: GTTAAACAAT GGCCATTGAC AGAAGA SEQ ID No. 20 5 rcversc: --

GGAATATTGC TGGTGATCCT TTCC SEQ ID No. 21 alternate t'orward: GTTAAACAAT GGCCATTGAC AG SEQ ID No. 22 1 () R'1'2 I'rimcrs t'orward: 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 t'orward: 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 t'orward primers are nested within the PR t'orvvard 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 dift'erent fluorescent dyes (such as, t'or example, the Visiblc Gcnetics Inc. MicroGene Clipper or l.ong-Read 'I'ower

-6 scquenccrs), hotly the forward and reverse primers arc prct'crably each labeled with one of the two dyes. Forward and rcvcrsc sequencing t'ragmcnts arc then generatcLI by thermally cycling the sample through multiple thermal cycles in the prcscncc ot'cithcr dtlA, dd l, ddC anal ddG. Analysis of the sequencing fragments produced using gel electrophorcsis 5 will allow the determination ot the positions of all 4 bases.

Finally, if the intermediate test tails to provide unambiguous identification ot the DNA type, sequencing ot'both strands may be performed. Again, the same sequencing primers identified above are used. I'orward and reverse sequencing fragments can be produced in a single reaction using distinctively labeled forward and reverse primers, or in 1() separate reactions depending on the nature ot'thc detection system being employed.

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 performing 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 dit'ferent region of the HIV- I genome, the pairs together Banking 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 pcrfonming t'our base sequencing may be included when intermediate and final assays on one strand and both strands are desired.

20 As used herein, the term "tcnnination mixture" ret'ers to a mixture containing a mixture of the t'our dcoxunucleotide triphosphatcs (dATI', dCTP, dG'l'P, and dTTP), one species of chain terminating didcoxynucleotidc (ddATP, ddCTP, ddGTP or ddTTP) and the appropriate sequencing primers.

The subkit t'or performing A and'l' seLIuencing on HIV-I may also be provided 25 separately t'or performing the initial determination of only the A and T nucleotides. A pret'crred kit of this type, whether provided separately or as part of a kit t'or pertonning a hierarchical assay has primer pairs in which each primer is labeled with a diticrent an spectroscopically distinguishahlc fluorescent dye, such as Cy5.() and Cy5. 5 and includes only one of the two possible types of termination mixtures, t'or example just the T 3() termination mixture.

The following examples arc included to illustrate aspects ot'thc instant invention and are not intended to limit the invention in any way.

( -7 EXAM['LE I

he RT-PC: R is done on the HIV- I RNA using a blend ot'en/.ymes fondling RT-PCR Master Mixes described below to conduct six R'l'-PC"I' reactions. 'I-his R'l'-l'CR 5 is done on the RNA preparation obtained using the QlAmp Viral RNA kit t'rom Qiagen. It can also he done on the I<NA extract for the NucliSense''' (t'onncrly known as NASBA) HIV Viral Load t'rom Organon'l'eknica.

All the reagents, tubes, tips, and other material needs to be RNase-t'ree.

10 'I'hc recipe is matte t'or 8 reactions (one strip ot'8 tubes), including 1 () /o extra. 'I'ha\v the RNA sample t'rom the Amplicor HIV Monitor Test and keep on ice. This is the material I obtained at step 14 ofthe section B "Specimen Preparation". It'using RNA prepared t'or the NucliSensc Assay, proceed the same way: thaw it and keep it on ice.

Take a 0.2 ml sterile, RNase-t'ree, centrit-'uge tube, RNase-free, and prepare the 15 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 1 of 80 mM DTT I 0.5 Ill of RNase-t'ree dNTP at I O mM each dNTP 2() 2 I Ill of forward PCR primer at 28 I1M.

21 Ill of reverse PCR primer at 28 EM 3.5 Ill ot'Rnase-inhibitor from Roche Molecular Biochemicals, catalog # 799 095 (1(),000 U) 25 Take a (). 2 ml sterile, RNase-free, centrit'uge tube, RNase-t'ree, and prepare the R'l'-PCR Master Mix 11 (enough for (j tubes) by adding the following ingredient in the order listed: RT-PCR MASTER MIX 11

7() Ill of 4x RT-PCR But'f'er (28() mM Tris Hcl, 3.2 mM MgCI., 6() mM (NH) ,SO4, 1 ()() 30 pa/ml Acetylated BSA from Life Tech, CA, pH 8.6() at 25"C) 3.5 al of RNase Inhibitor at40 U/,ul 7 Ill of Superscript 11

( -8 .75 Ill of F.xpand High lidelity Enzymc Systcm F:nzymc Mix from Roche Molecular 13iochemicals, catalog h 1733381X S.75 Ill ol'AmpliTaq t'rom Rochc Molecular Systems.

I'akc one strip of 6 thin wall tubes. Add 9 Ill ot'MASTER MIX I iT1 each tube.

5 Add 17 1 ot'sample (RNA) to each tube. You may want to add a negative control per experiment. Ileat the RNA sample at 9()0C tor 2 min. using the program below:, cool at 50"C and add 14 1 ofthe MASTER MIX 11 in each tube (step 3 ot'thc program below). Be careful not to cross contaminate your samples.

Start the RT-PCR. Use the heated lid. When using the MJ-Plates, indicate I () that tubes arc used when asked by the P'FC-20(). The tollowin,g is the programming for the PTC-200:

Calculated I = 90.0 for ever 2= 90.0 t'or 2:00 15 3= 50.0 for 1:00:00 4= 94.0 for 2:0() 5= 1.0 /s to 94.0 6= 94.0" for 0:30 7= 1.0 /s to 57.0 20 a= 57.0 for 0:3() 9= 1.0''/s to 68.()0 10= 68.0' t'or 2:00 I 1=Goto 5, 19 times 12= 1.()"/s to 94.0 25 13= 94.0" for 0:3() 14= 1.0"/s t<'60.0 15 - 60.() for ():3() 1 6= 1.0 /s try 68.0'' 17= 68.0'' t'or 2:30 30 18=Goto 12, 16times 19= 68.0' t'or 7:00 2()= 4.0 for ever

- - 21 --End I-XAMPLI-. 2

TO determine the sequence ot'arnplicon, 7 Ill of each terminator mix (16 when 5 using a two dyc instrument) arc combined with a 5 ul ot'a master mix as'tllows: MASTER MIX (two-dye system) t'or 6 tubes, i.e. tor 6 samples: 12() 111 t,t'hut't'cr (26() mM 'I'ris-HC'I, pH 8.3 at 25"C, 32.5 mM M,CI) 475 Ill ot'stcrile watcr 22.5 Ill enzyme blend of AmpliTacl FS t'.om Roche Molecular Systems 15 U/lll and 27 I O I)/1 pyrophosphotase 5 Ill of the PCR product t'rom Example 3 per tube.

The two mixtures are mixetl ently with a pipette tip, and the thermocylcing reaction is started. The t'ollowing is the programming for thc PTC-200: Calculated 15 1= 94.0 for 5:00 2= 1.0 /s to 94.0 3= 94.0 tor 0:20 4= 1.0/s t, 56.0" 5--- 56.0 tor 0:20 2() 6-- 1.0"/s to 70.0 7= 70.() for 1:3() 8=Goto 2, 29 times 9= 70.0''to,r 5:00 10- 4.() tc,rcver 25 11=End I ermination mixes t'or two dye systems Proteasc A-Mix: 1.07 I1M ddATP; 643 I1M dATP; 643 I1M dC"l'P; 643 I1M dG'I'P; 643 M dTTP; 30 330 nM total of forward primers and 33() nM to,tal of reverse primers; I mM Tris-HCI, pH 8.0 at 25"C, 0.1 mM ED'I'A.

C-Mix: 2.14 I1M ddCl'P; 643 I1M dATP; 643 M dCTP; 643,uM dGTP; 643,uM dTTP;

-1O- -

33() nM t<'tal of forward primers and 330 nM total ot'rcversc primers; I mM Tris-l ICI, pFJ S.U at 25"C, (). MnM EDTA.

G-Mix: 2.14 I1M ddGTP; 643,uM clATP 643 M dCTP; 643 I1M dCi'l'P; (43 I1M tlTTP; 33() nM total ot'forward primers ancl 33() nM total of rcvcrsc primers; I mM '1'ris-HCI, pil 5.() at 25"C, (). I mM EDTA.

T-Mix: 2.14 M ddTTP; 643 M dA'1'1'; 643 M dC'1'1'; 643 M dGTP; 643 M d'l"l'l'; 33() nM total of f'orward primers and 330 nM total ot' reverse primers; I mM 'I'ris-l IC'I, pH S.() at 25 C, (). I mM EDTA.

Both primers are labeled, tor example with CyS.O and CyS.5, respectively.

First RT region A-Mix: 1.07 M ddATP; 643,uM dATI'; 643 M dCl'P; 643 I1M dG'l'P; 643 M dTTP; 330 nM total of forward primers and 330 nM total of reverse primers; I mM Tris-HCI, pH S.O at 25 C, 0. I mM EDTA.

15 C-Mix: 2.14,uM ddCTP; 643 M dATP; 643 I1M dCTP; 643 I1M dGTP; 643 M dTTP; 330 nM total of t'orward primers and 330 nM total of reverse primers; I mM Tris-HCl, pH S.0 at 25"C, 0. I mM EDTA.

G-Mix: 2.14 M ddGTP; 643,uM dATP; 643 I1M dCTP; 643,uM dGTP; 643 I1M dTTP; 330 nM total of forward primers and 330 nM total ot'revcrse primers; I mM Tris-HCI, pl I 2() t3.0 at 250C, 0.1 mM EDTA.

T-Mix: 2.14 I1M ddTTP; 643 M dAl'P; 643 I1M dC'l'P; 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 CyS.O and Cy5.5, respectively.

25 Second reverse transcriptase region A-Mix: 1.()7 I1M ddATP; 643 M clAl'P; 643 llM dC'I'P; 643 M d(GTP; 643 I1M dTTP; 33() nM total of forward primers and 330 nM uta1 of reverse primers C-Mix: 2.14 I1M ddCTP; 643 I1M dATP; 643 M dCEP; 643 I1M dG'1'1'; 643 I1M dl'TP; 330 nM total of forward primers and 330 nM total of reverse primers 30 G-Mix: 2.14 I1M ddGTP; 643 M dATP; 643,uM dCTP; 643 I1M dG'l'P; 643 I1M dTTP; 330 nM total of t'orward primers and 33() nM total of reverse primers T-Mix: 2. 14 I1M dd'l'TP; 643,uM dATP; 643 I1M dCTP; 643,uM dGTP; 643 M dTTP;

( - 33() nM total of fovarLI primers and 33() nM total ot'rewerse primers E3oth primers arc labclcd, for example with ('y5 () and C'y5 5, respectively P2 protease region 5 A-Mix 1 ()7 I1M ddATT,; 643 M dATP; 643 I1M d("l'P; 643 I1M dGTI', 643 I1M dTTP; 330 nM total ot'forward primers and 33() nM total of reverse primers C-Mix 2 14 I1M ddCTP; 643 I1M dATP; 643,uM dCTP; 643 I1M d(i'l'P; (43 M dTTP; 330 nM total of forward pimers and 33() nM total of reverse primers G-Mix 2 14 M ddGTP; 643 I1M dA'l'P; 643 I1M dC'l'P; 643 M dGTP; 643 M dTTP; 10 330 nM total ol' forward primers and 330 nM total of reverse primers T-Mix 2 14 I1M dd'TI'; 643 1M dATP; 643 M dCTP; 643 I1M dGTP; 643 I1M dTTP; 33() nM total of forward primers and 330 nM total of reverse primers Both primers are labeled, for example with Cy5 () and Cy5 5, respectively

ILL SEQUENCE LISTING

a_ <110> Visible Genetics Inc <120> Method And Kit For Evaluation Of HIV Mutations <130> P450237

<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 c400> 1 aagcaggagc cgatagacaa gg 22 c210> 2 c211> 22 c212> DNA c213> 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

c213> Human immunodeficiency virus <400> 4

ctaytargtc ttttgwtggg tcata 25 c210> 5 <211> 24

<212> DNA

c213> Human immunodeficiency virus <400> 5

gctattaagt ctthUgatgg geca <210> 6

<211> 24

<212> DNA

<213> Human immunodeficiency virus <400> 6

gagacratag acaaggaayt rtat 24 <210> 7

c211> 24 <212> DNA

<213> Human immunodeficiency virus <400> 7

gagmcgatag acaaggrvct gLat 24 <210> 8

<211> 21

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<213> Human immunodeficiency virus <400> 8

achtttgggc catccattcc t 21 <210> 9

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<212> DNA

<213> Human immunodeficiency virus <400> 9

gagccgatag acaaggaact atatcc 26 <210> 10

<211> 26

<212> DNA

c213> Human immunodeficiency virus <400> 10

1: gagccgatag acaaggeagt atatcc26 <210> 11I

<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

c212> DNA <213> Human immunodeficiency virus <400> 13

gagccgatag acaaggaagt gtatcc26 <210> 14

<211> 26

<212> DNA

<213> Human immunodeficiency virus <400> 14

gagccgatag acaaggaaat gtatcc26 <210> 15

<211> 26

<212> DNA

<213> Human immunodeficiency virus <400> 15

gagccgatag acaagggact gLatcc26 <210> 16

<211> 26

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<213> Human immunodeficiency virus <400> 16

gagocgatag acaaggacat gtatcc26 1 A- <210> 17

c211> 26 c212> DNA c213> Human immunodeficiency virus c400> 17 gagccgatag acaagggcat gtatcc 26 c210> 18 c211> 26 c212> DNA c213> Human immunodeficiency virus <400> 18

gagacgatag acaaggagct gLatcc26 c210> 19 <211> 26

<212> DNA

<213> Human immunodeficiency virus <400> 19

gagccgatag acaaggggct gtatcc26 <210> 20

<211> 26

<212> DNA

<213> Human immunodeficiency virus <400> 20

ghtaaacaat ggacattgac agaaga26 <210> 21

<211> 24

<212> DNA

<213> Human immunodeficiency virus <400> 21

ggaatattgc tggtgatcct tCcc24 <210> 22

<211> 22

<212> DNA

<213> Human immunodeficiency virus

<400> 22

ghtaaacaat ggccattgac ag 22 <210> 23

c211> 27 <212> DNA

<213> Human immunodeficiency virus <400> 23

gaagLatact gcathtacca tacctag 27 <210> 24

<211> 27

<212> DNA

<213> Human immunodeficiency virus <400> 24

gaagtatact gcathtacta tacctag <210> 25

<211> 27

<212> DNA

<213> Human immunodeficiency virus <400> 25

aaagtatact 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

tchgLaLgtc attgacagtc 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

tctgtatatc attgacagtc cage 24 <210> 30

<211> 25

<212> DNA

<213> Human immunodeficiency virus <400> 30

ttchgtaLgt cattgacagt ccagc 25 <210> 31

<211> 21

<212> DNA

<213> Human immunodeficiency virus <400> 31

ttccatcaga tcactctttg g 21 <210> 32

<211> 21

<212> DNA

<213> Human immunodeficiency virus <400> 32

ttccctcaaa tcactctttg g 21 <210> 33

<211> 21

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acthttgggc catccatecc t 21

Claims (1)

1. to Claims:

   I. A method for determining the genetic type of HIV-I present in a sample containing HIV-I, comprising determining the positions of the nucleotides within the protease 5 gene and comparing these positions to the positions of nucleotides in known genetic types, using at least one primer consisting of SEQ ID No's 8 or 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 lo 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.

   5 4. A kit for performing sequencing on an HIV-I genome, comprising a plurality of termination mixtures, each of said termination mixtures including one of a plurality of primerpairs, each pair flanking a different region ofthc HIV-1 genome, and at least one member of each pair being labelled with a detectable label, wherein the primers are selected from the group consisting of: 20 a primer pair for sequencing a portion of the protease gene comprising a forward primer selected from the group consisting of SEQ ID No's 6, 7, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18 and 19, and a reverse primer having the sequence of SEQ ID No 8; or a primer pair for sequencing a portion of the protease gene comprising a forward primer selected from the group consisting of SEQ ID No's 31 and 32, and a reverse primer 25 having the sequence of SEQ ED No 33; and 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.