GB2395009A

GB2395009A - Method and kit for evaluation of HIV mutations

Info

Publication number: GB2395009A
Application number: GB0402069A
Authority: GB
Inventors: James M Dunn; Jean-Michel Lacroix
Original assignee: Visible Genetics Inc
Current assignee: Visible Genetics Inc
Priority date: 1999-10-15
Filing date: 2000-09-20
Publication date: 2004-05-12
Anticipated expiration: 2020-09-20
Also published as: GB2395007A; GB0402062D0; GB0402069D0; GB2395008B; GB2395008A; GB0402064D0; GB2395009B

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

i MF.TI IOD AND KIT FOR l'2VAL.UA'LlON OF HIV MIJTA'I'IONS BAC'K(-,ROlJNO

OF'I'HF. INVENTION Genctic testing to determine the presence ot'or a susceptibility to a disease condition ol'f'ers incredible opportunities for improved medical care, and.tlle potential for 5 such testing increases almost daily as ever increasing numbers ot'disease-associated genes and/or mutations arc 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 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 I O 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 confommational polymorphism" ( "SSCP") described by 15 Orita et al., Cenomics 5: 874-879 ( 1989), or by a modification thereof referred to a dideoxy-fingerprinting ("ddF") described by Sarkar et al., C,e,omics 13: 441()443 (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 ot'the size ot'the t'ragTnents and of the three-dimensional 90 cont'onnation of the undenatured t'ragments. 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 (lIIV). SUMMARY OF TIIE INVENTION

The method of the invention provides a method for obtaining int'onnation about the allelic type of a sample of genetic material derived t'rom an lllV-intected sample. A test is pert'onned in which the sequence is determined in the 3'-direction t'or all 3() 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 t'or all four bases.

To pcrtonn the method of the invention, reagents suitable tor the tests are suitably packaged as a kit. I he kit contains reagents for pcrtonning a tour-base sequence determination on one or both strands ot the target DNA. Onc-strandcd scqucncc determination could be performed all in the 3'dircction, all in the 5'-dircction, OT as a 5 combination ot the two strands.

BRIM DESCRIPTION Ol THE DRAWINGS

Fig. I shows a schematic representation of the invention.

10 DETAILED DESCRIPTION Ol: THE INVENTION

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

1. "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 tour 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 l'ublication No. WO 97/23ti5(), certain alleles of a genetic locus may be distinguishable On the basis ot'idcntit'ication of the location ot less than four' and often only one nucleotide. This tending allows the cIcvelopment ot'the present method For improved allele identification within the highly polymorphic HIV genome.

s 'I'raditionally, it'sequencing were going to be used to evaluate the allelic type ot'a polymorphic gene, four dideoxy nuclcotide "sequencing" reactions of the type described by Sanger et al. (Proc. Natl. Acad. Sci. t]SA 74: 5463-54(i7 ( 1')77)) wouLl be run on the sample concurrently, and the products of the tour reactions would then be analyzed by polyacrylamide gel electrophoresis. (see Clip 7.6, Cunrent Protocols in 10 Molecular Biology, Eds. Ausubel, I:.M. ct al. (John Wiley & Sons; 19') 5)) In this well known technique, each of the Our sequencin, 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 ot'base in the extension product, but does not reveal the order and type of nucleotides intervening between the I 5 bases of this specific type. The information provided by the tour 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 bet'ore 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 OpenGene''M System: the MicroGene Clippers or I.ong-Read 25 Tower_ 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 protease and reverse transcriptase genes of the HIV genome from a target cell. This reaction can be 3() performed using, for example, the TITAN_ One-Tube RT-PCR system from Boehringer Mannbeim (Cat. No. 1 855 47fi or 1 882 382) using the following primers: forward primer set:

( 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't'er (260 mM Tris-HCL, pH 8.3; 32.5 mM Mg,C'I, at 25 "C) and a polymerase enzyme I () such as Taq FS (Perkin Elmer/Applied Biosystems (at No. 4()907()) This polymerase has a high rate of incorporartion of dideoxynucleotide relateive to the incorporation rate ot; for example, conventional Taq polymerase. This mixture is used as stock in the subsequent reactions. 1'he sequence reaction is pertorrned on the protease gene using the t'olkwing 1 5 primers: t'orward 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 he 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 revcrsc transcriptase pcnc, three sets ot'primcrs arc used as follows: R'l'l Primers rward: GTTAAACAAT GGCCATTGAC AGAAGA SEQ ID No. 20 5 reverse: -

GGAATATTGC TGGTGATCCT TTCC SEQ ID No. 21 alternate t'orwarti: GTTAAACAAT GGCCATTGAC AG SEQ ID No. 22 1(1 RT2 I'rimers 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 forward: 25 TTCCCTCAGA TCACTCTTTG G SEQ ID No. 31 TTCCCTCAAA TCACTCTTTG G SEQ ID No. 32 revcrsc: ACTTTTGGGC CATCCATTCC T SEQ ID No. 33 30 The 1'2 forward primers are nested within thc PR forward primers to sequence samples which do not sequence with the PR primers. When a sequencing device is cmploycd which is capable of detecting and distinguishing two different fluorescent dyes (such as, t'or example, the Visible Genetics Inc. MicroGene Clipper or Long-Read Tower

-6- sequencers), troth the forward and reverse primers are pret'crably each labticd with one ot the two dyes. Forward find reverse sccluencing fragments are then gentratctl by themllly cycling the sample through multiple teal cycles in the presence ot'either ddA, ddT, ddC and ddG. Analysis of the sequencing t'ragments produced using gel electrophorcsis 5 will allow the determination of the positions of all 4 hascs. -

l inally, if the intermediate test tails to provide unambiguous identification of the DNA type, sequencing ot'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 1() separate reactions depending on the nature ot'the detection system being employee.

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 ot' an HIV-I gene in a sample comprising: a kit t'or performing t'our base sequencing on HIV 1 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 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.

20 As used herein, the tend "termination mixture" rettrs to a mixture containing a mixture of the four deoxunuclcotide triphosphates (dATP, dCTP, dG'l'P, and dTTP), one species of chain terminating dideoxynucleotide (ddATI,, ddCTP, ddGTP or ddT'l'P) and the appropriate sequencing primers.

The subkit for pert'orrning A and T sequencing on HIV-I may also be provided 25 separately t'or pert'orming the initial determination openly the A and T nucleotides. A pret'erred kit of this type, whether provided separately or as part ot'a kit t'or pert'onming a hierarchical assay has primer pairs in which each primer is labeled with a tlit't'erent an spectroscopically distinguishable fltrorescent dye, such as Cy5.() and CyS.5 and includes only one of the two possible types ot'tennination mixtures, t'or example just the T 30 termination mixture.

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

-7 __ F.XAMI'LI'. I

Thc RT-I'CR is done on the HIV- I IlNA using a blend of enzymes forming, RT-PCR Master Mixes described below to conduct six R'l'-r'C1' reactions. This RT-I'C'R 5 is done on the RNA preparation obtained using the (,?lAmp Viral R>IA Lit from Qiagen. It can also be done on the RNA extract for the NucliSense''M (tonncrly kno\vn as NASE3A) HIV Viral Load from Organon Teknica.

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

1() Thc recipe is made t'or reactions (one strip ot'S tubes), includin, I()/o 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 tor the NucliSense Assay, proceed the same way: thaw it and keep it on ice.

Take a 0.2 ml sterile, RNasc-t'ree, centrifuge tube. RNase-free, and prepare the I 5 RT-PCR Master Mix I (enough for 6 tubes, i.e. 6 samples) by addin, the following ingredient in the order listed: RT-PCR MASTER MIX I

7 pi ot'S0 mM DT1' 10.5 Ill of RNase-t'ree dNTP at 10 mM each dNTP 20 21 pi 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 Biochcmicals, catalog 799 025 (1(),000 U) 25 Take a ().2 ml sterile, RNasc-tee, centritige tube. RNasc-t'ree, and prepare the R'l'l'CR Master Mix II (cnougl1 t'or 6 tubes) by adding the following ingredient in the order listed: RT-PCR MASTER MIX 11

7() PI of 4x R'l'-PCR But't'er (2XO mM Tris Hcl, 9.2 mM MgCI,, 60 mM (N114).SO;, 1()0 30,ug/ml Acctylated BSA t'rom bite Tech, CA, pH S.60 at 25"C) 3.5 pi of RNase Inhibitor at4() U/pl 7 Ill of Superscript 11

S.75 al ot Expand High Fidelity F.nzyme System Enzyme Mix from Roche Molecular 13iochemicals, catalog # 17333 8 I X S.75 Ill of AmpliTaq from Roche Molecular Systems.

Fake one strip of 6 thin wall tubes. Add 9 Ill ot MASTER MIX I in each tube.

5 Add 17 Ill ot sample (RNA) to each tube. You may want to add a negative control leer experiment. Heat the RNA sample at 9() C tor 2 min. using the program below:, cool at 5() C and add 14 Ill of the MAS I F.R MIX 11 in each tube (step 3 ot the program below). Be careful not to cross contaminate your samples.

Start the R l -PCR. Use the heated lid. When using the MJ-Plates, indicate I () that tubes are used when asked by the PTC-200. I he iollowiilg is the programming for the PTC-200:

Calculated I = 90.0 for ever 2= 90.0' tor 2:00 15 3= 50.0 tor 1:00:00 4= 94.0 for 2:00 5= 1.0 /s to 94.0 6= 94.0 for 0:30 7= 1.() /s to 57.0 20 8= 57.0 for 0:30 9= 1.0 /s to 68.0 10= 63.0 tor2:00 11 =Goto 5, 19 times 12= 1.0 /s to 94.0 25 13= 94.() torO:3() 14-- 1.() /s to 60.0 15= 60.0 for 0:30 16= 1.0 /sto 68.0 17= 68.0 for 2:30 30 18=Goto 12, 16 times 19= 68.0' for 7:00 20= 4.0 for ever

( - 2 I - Enl EXAMrl F.2 To detcnninc the setlucncc of anplicon, 7 Ill of each tenninator mix (16 when 5 using a two dye instrument) are combined with a 5 ul ot'a master mix as t'ollows: MASTER MIX (two-dye system) for 6 tubes, i.e. tor 6 samples: I 2() Ill of but'ter (26() mM Tris-HCI, pH 8. 3 at 25"C 32.5 nM MC: I.) 475 Ill ot'sterilc water 22.5 Ill enzyme blend ol'Ampli'l'aq F5i t'rom 1tochc Molecular Systcms 15 U/1 and 27 I () [J/lll pyrophosphotase 5 Ill of the PCR product from F.xamplc 3 per tube.

I'he two mixtures arc mixed gently with a pipette tip, and the thermocylcing reaction is started. 'I'he tollowin:, is the programTning for the PTC-200: Calculated 15 1= 94.()'' fUr 5:00 2-- 1.0 /s to 94.0 3= 94.() t'or 0:2() 4= 1.0 /s to 56.00 5= 56.() for 0:() 20 6= 1.0/sto 70.0 7- 70.()" for 1:30 8--Goto 2, 29 times 9= 70.0 for 5:0() 10-- 4.() t'or ever 25 11 --End Tcnnination mixes for two dye systems Protease A-Mix: 1. ()7 I1M ddATP; 643 I1M dATP; 643 M dC'l'P; 643 I1M dGTY; 643 I1M dTTP; 30 330 nM total of t'orward primers and 33() nM total ot reverse primers; I mM Tris-HCI, pH 8.() at 25"C, (). I mM EDTA.

C-Mix: 2.14 I1M ddCTP; 643 I1M dATP; 643 M dCTP; 643,uM dGTP; 643 I1M dTTP;

i -10 33() nM total ot't'orward primers and 33() nl total ot'rcvcrsc primers; I mM Tris-HCI, pH 8.0 at 95"C', ().1 mM EDTA.

G-Mix: 2.14 I1M ddG'I'P; 643 I1M dA'I'I'; 643 I1M dC'l'P; 643 M dGTP; 643 M d'l'TP; 33() nM total of forward primers and 330 nM total ot'rcvcrse primers; I mM Tris-HC'I, pl I 5 8.0 at 95"C, 0.1 mM F.DTA. --

1'-Mix: 2.14 I1M ddTTP; 643,uM dATP; 643 M dCl'P; 643 M dGTP; 643 M d'l''l'l'; 33() nM total of t'orward primers and 33() nM total of rcvcrse primers; I mM 'I'ris-l-lC'I. pH 8.0 at 25 C, 0.1 mM ED'I'A.

Both primers are labeled, for example with Cy5.0 and Cy5.5, respectively.

1() First R'l' region A-Mix: 1.07 1M ddATP; 643 M dATP; 643 I1M dCTP; 643 I1M dGTP; 643 I1M dTTP; 330 nM total of t'orward primers and 330 nM total ot'rcverse primers; I mM 'I'ris-HCI, pH 8.0 at 25"C, 0. I mM EDTA.

15 C-Mix: 2.14 I1M ddCTP; 643 I1M dATP; 643 M dCTP; 643 M dG'l'P; 643 M dT'l'P; 330 nM total of t'orward primers and 330 nM total ot' reverse primers; I mM Tris-HCI, pH S.0 at 25"C, 0.1 mM EDTA.

G-Mix: 2.14 I1M ddGTP; 643 M dATP; 643 I1M dCTP; 643 I1M dGTP; 643 M dTTP; 330 nM total of krward primers and 330 nM total of reverse primers; I mM Tris-HCI, pi-l 2() 8.0 at 25"C, 0.1 mM EDTA.

T-Mix: 2.14 M ddTTP; 643 '1M dATP; 643 M dCTP; 643,uM dGTP; 643 M dTTI'; 330 nM total of t'orward primers and 330 nM total of reverse primers Both primers are labeled, for example with Cy5.0 and Cy5.5, respectively.

25 Second reverse transcriptase region A-Mix: 1.()7 M ddATP; 643 I1M dATP; 643 I1M dCTP; 643 I1M dGTP; 643,uM d'l'TP; 33() nM total of forward primers and 330 nM total ot'reverse primers C-Mix: 2.14 I1M ddCTP; 643 I1M dATP; 643 M dCTP; 643 I1M dGTP; 643 I1M d'l'TP; 330 nM total ot' forward primers and 33() nM total ot' reverse primers 30 G-Mix: 2.14 I1M ddGTP; 643 I1M dATI'; 643 I1M dCTP; 643 I1M dGTP; 643 M dTTP; 330 nM total of forward primers and 330 nM total of reverse primers T-Mix: 2.14 I1M ddT'l'P; 643 I1M dATP; 643 M dCTP; 643 I1M dGTP; 643 I1M d'l'TP;

-1 1 33() nM total ot'tor\vard primers and 33() nM total ot'rewersc primers 130th primers arc labeled, for cxampic witlh (.y5.() and (:'yS.5, rcspcctively.

P2 protcasc rcion 5 A-Mix: 1.()7 I1M dtlATP; 643 I1M dATP; 643,uM d(:'l'l'; (>43 M dG'l'P, 643 M dTTP; 330 nM total ot't'orward primers and 33() nM total ot'reverse primers C-Mix: 2.14 I1M ddC'1'1'; 643 I1M dATP; 643 I1M dC''I'I': (i43 M dG'l'P; 643 I1M dT'I'P; 330 nM total of t'orward primers and 330 nM total ot'reverse primers G-Mix: 2.14 I1M ddGTP; 643 I1M dATP; 643,uM dCTP; 643 M dC,TP; 643 M dTTP; 10 330 nM total ot't'orward primers and 330 nM total ot'reversc primers T-Mix: 2.14 M dlTTP; 643 I1M dATP; 643 M dC'l'P; 643 I1M dGTP; 643 M dTTP; 330 nM total ot'tor\vard primers and 330 nM total ot'revcrse pimers Both primers are labeled, tor example with Cy5.() and Cy5.5, respectively.

1) SEQUENCE LISTING

a_ <110> Visible Genetics Inc <120> Method And Kit For Evaluation Of HIV Mutations <130> P450287

<140> <141> <150> US O9/418720

<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 thttgweggg tcata 25 <210> 5

<211> 24

<212> DNA

<213> Human immunodeficiency virus c400> 5 gctattaagt ctthtgaLgg gtca 24 <210> 6

<211> 24

<212> DNA

<213> Human immunodeficiency virus <400> 6

gagccratag acaaggaayt rtat 24 <210> 7

<211> 24

<212> DNA

c213> Human immunodeficiency virus <400> 7

gagmcgatag acaaggrvct Stat 24 <210> 8

<211> 21

c212> DNA c213> Human immunodeficiency virus c400> 8 actthtgggc catccathac t 21 <210> 9

<211> 26

c212> DNA <213> Human immunodeficiency virus <400> 9

gagccgatag acaaggaact atatcc 26 <210> 10

c211> 26 c212> DNA <213> Human immunodeficiency virus c400> 10

1 l_,, gagccgatag acaaggaagt atatcc26 <210> 11-

<211> 26

c212> DNA <213> Human immunodeficiency virus c400> 11 gagccgatag acaaggeaat atatcc 26 <210> 12

<211> 26

<212> DNA

c213> Human immunodeficiency virus <400> 12

gagcagatag acaaggaact gtatcc26 c210> 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

gagcagatag acaaggaaat gLatcc26 <210> 15

<211> 26

<212> DNA

<213> Human immunodeficiency virus <400> 15

gagcogatag acaagggact gtatcc26 <210> 16

<211> 26

<212> DNA

<213> Human immunodeficiency virus <400> 16

gagacgatag acaaggacct gtatcc26 A_ <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

gagacgatag acaaggagct gLatcc26 <210> 19

<211> 26

<212> DNA

<213> Human immunodeficiency virus <400> 19

gagcogatag acaaggggct gtatcc26 <210> 20

<211> 26

<212> DNA

<213> Human immunodeficiency virus <400> 20

gttsaacaat ggccattgac agaaga26 <210> 21

<211> 24

<212> DNA

<213> Human immunodeficiency virus <400> 21

ggaatattgc tggtgatcct ttcc24 <210> 22

<211> 22

<212> DNA

<213> Human immunodeficiency virus

<4uO> 22 ghtaaacaat ggccattgac ag 22 <210> 23

<211> 27

<212> DNA

<213> Human immunodeficiency virus <400> 23

gaagLatact gcatetacca tacctag 27 <210> 24

<211> 27

<212> DNA

<213> Human immunodeficiency virus <400> 24

gsagLatact gcatttacta tacatag 2/ <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 gcatUtacca tayctag 27 <210> 27

<211> 24

<212> DNA

<213> Human immunodeficiency virus <400> 27

tctgtatgtc attgacagec cage 24 <210> 28

<211> 24

<212> DNA

<213> Human immunodeficiency virus

1 ? <400> 28

tctgLatatc 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

tCctgLaLgt cattgacagt ccagc 25 <210> 31

<211> 21

<212> DNA

<213> Human immunodeficiency virus <400> 31

tCcactcaga 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

actettgggc catccathac t 21

Claims

)& Claims:

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 protease 5 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 31 and 32.

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 HIV-I 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 ofthe HIV- I genome, and at least one member of each pair being labelled with a detectable label, wherein the primers are 20 sclcctcd from the group consisting of: a primer pair for sequencing a portion ofthe protease gene comprising a forward primer selected from the group consisting of SEQ ID No's 31 and 32, and a reverse primer having the sequence of SEQ ID No 33; or 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.