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:
[0001]    This application is a continuation-in-part of U.S. patent application Ser. No. 08/938,641, filed Sep. 26, 1997, U.S. patent application Ser. No. 08/577,858, filed Dec. 22, 1995, and U.S. patent application Ser. No. 08/819,912 filed Mar. 18, 1997, which are incorporated herein by reference. 
     
    
     
       BACKGROUND OF THE INVENTION  
         [0002]    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.  
           [0003]    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.  
           [0004]    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 INVENTION  
         [0005]    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.  
           [0006]    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 combination of the two strands. 
       
    
    
     BRIEF DESCRIPTION OF THF DRAWINGS  
       [0007]    [0007]FIG. 1 shows a schematic representation of the invention. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0008]    While the terminology used in this application is standard within the art, the following definitions of certain terms are provided to assure clarity.  
         [0009]    1. “Allele” refers to a specific version of a nucleotide sequence at a polymorphic genetic locus.  
         [0010]    2. “Polymorphic site” means a given nucleotide location in a genetic locus which is variable within a population.  
         [0011]    3. “Gene” or “Genetic locus” means a specific nucleotide sequence within a given genome.  
         [0012]    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.  
         [0013]    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.  
         [0014]    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          gation 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 97/23,650, 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.  
         [0015]    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 &amp; 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 arrive at a composite picture of the sequence as a whole.  
         [0016]    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 OpenGene™ System: the MicroGene Clipper™ or Long-Read Tower™ DNA Sequencers, GeneObects™ and GeneLibranian™ Software.  
         [0017]    [0017]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 TITAN™ 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          
 
         [0018]    This amplicon is then combined with a master sequencing mixture containing buffer (260 mM Tris-HCL, pH 8.3; 32.5 mM MgCl 2  at 25° C.) and a polymerase enzyme such as Taq FS (Perkin Elmer/Applied Biosystems Cat No. 402070) This polymerase has a high rate of incorporation 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:                GAGCCGATAG ACAAGGAACT ATATCC   SEQ ID No. 9               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           CAGCCGATAG 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 uanscriptase gene, three sets of           primers are used as follows:                RT1 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 C   SEQ ID No. 31           TTCCCTCAAA TCACTCTTTG C   SEQ ID No. 32                       reverse:           ACTTTTGGGC CATCCATTCC T   SEQ ID No. 33          
 
         [0019]    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 forward 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.  
         [0020]    Finally, if the intermediate test ails 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.  
         [0021]    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.  
         [0022]    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.  
         [0023]    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.  
         [0024]    The following examples are included to illustrate aspects of the instant invention and are not intended to limit the invention in any way.  
       EXAMPLE 1  
       [0025]    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 NucliSense™ (formerly known as NASBA) HIV Viral Load from Organon Teknica.  
         [0026]    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.  
         [0027]    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:  
         [0028]    RT-PCR MASTER MIX I  
         [0029]    7 μl of 80 mM DTT  
         [0030]    10.5 μl of RNase-free dNTP at 10 mM each dNT1  
         [0031]    21 μl of forward PCR primer at 28 μM.  
         [0032]    21 μl of reverse PCR primer at 28 μM  
         [0033]    3.5 μl of Rnase-inhibitor from Roche Molecular Biochemicals, catalog#799 025 (10,000 U)  
         [0034]    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:  
         [0035]    RT-PCR MASTER MIX II  
         [0036]    70 μl of 4×RT-PCR Buffer (280 mM Tris HCl, 9.2 mM MgCl 2 , 60 mM (NH 4 ) 2 SO 4 , 100 μg/ml Acetylated BSA from Life Tech, CA, pH 8.60 at 25° C.) 
         [0037]    3.5 μl of RNase Inhibitor at 40 U/μl  
         [0038]    7 μl of Superscript II  
         [0039]    8.75 μl of Expand High Fidelity Enzyme System Enzyme Mix from Roche Molecular Biochemicals, catalog# 17333 818  
         [0040]    8.75 μl of AmpliTaq from Roche Molecular Systems. 
         [0041]    Take one strip of 6 thin wall tubes. Add 9 μl of MASTER MIX I in each tube.  
         [0042]    Add 17 μl of sample (RNA) to each tube. You may want to add a negative control per experiment.  
         [0043]    Heat the RNA sample at 90° C. for 2 min. using the program below: cool at 50° C. and add 14 μl of the MASTER MIX II in each tube (step 3 of the program below). Be careful not to cross contaminate your samples.  
         [0044]    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:  
         [0045]    Calculated  
         [0046]    1=90.0° for ever  
         [0047]    2=90.0° for 2:00  
         [0048]    3=50.0° for 1:00:00  
         [0049]    4=94.0° for 2:00  
         [0050]    5=1.0°/s to 94.0° 
         [0051]    6=94.0° for 0:30  
         [0052]    7=1.0°/s to 57.0° 
         [0053]    8=57.0° for 0:30  
         [0054]    9=1.0°/s to 68.0° 
         [0055]    10=68.0° for 2:00  
         [0056]    11=Goto 5, 19 times  
         [0057]    12=1.0°/s to 94.0° 
         [0058]    13=94.0 ’ for 0:30  
         [0059]    14=1.0°/s to 60.0° 
         [0060]    15=60.0° for 0:30  
         [0061]    16=1.0°/s to 68.0° 
         [0062]    17=68.0° for 2:30  
         [0063]    18=Goto 12, 16 times  
         [0064]    19=68.0° for 7:00  
         [0065]    20=4.0° for ever  
         [0066]    21=End  
       EXAMPLE 2  
       [0067]    To determine the sequence of amplicon, 7 μl of each termination mix (16 when using a two dye instrument) are combined with a 5 Ul of a master mix as follows:  
         [0068]    MASTER MIX (two-dye system) for 6 tubes, i.e. for 6 samples:  
         [0069]    120 μl of buffet (260 mM Tris-HCl, pH 8.3 at 25° C., 32.5 mM MgCl 2 )  
         [0070]    475 μl of sterile water  
         [0071]    22.5 μl enzyme blend of AmpliTaq FS from Roche Molecular-Systems 15 U/μl and 27 U/μl pyrophosphotase  
         [0072]    5 μl of the PCR product from Example 3 per tube.  
         [0073]    The two mixtures are mixed gently with a pipette tip, and the thermocycling reaction is started. The following is the programming for the PTC-200:  
         [0074]    Calculated  
         [0075]    1=94.0° for 5:00  
         [0076]    2=1.0°/s to 94.0° 
         [0077]    3=94.0° for 0:20  
         [0078]    4=1.0°/s to 56.0° 
         [0079]    5=56.0° for 0:20  
         [0080]    6=1.0°/s to 70.0° 
         [0081]    7=70.0° for 1:30  
         [0082]    8=Goto 2, 29 times  
         [0083]    9=70.0° for 5:00  
         [0084]    10=4.0° for ever  
         [0085]    11=End  
         [0086]    Termination mixes for two dye systems  
         [0087]    Protease  
         [0088]    A-Mix: 1.07 μM ddATP; 643 μM dATP; 643 μM dCTP; 643 μM dGTP; 643 μM 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.  
         [0089]    C-Mix: 2.14 μM ddCTP; 643 μM dATP; 643 μM dCTP; 643 μM dGTP; 643 μM 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.  
         [0090]    G-Mix: 2.14 μM ddGTP; 643 μM dATP; 643 μM dCTP; 643 μM dGTP; 643 μM 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.  
         [0091]    T-Mix: 2.14 μM ddTTP; 643 μM dATP; 643 μM dCTP; 643 μM dGTP; 643 μM dTTP;  
         [0092]    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.  
         [0093]    Both primers are labeled, for example with Cy5.0 and Cy5.5, respectively.  
         [0094]    First RT Region  
         [0095]    A-Mix: 1.07 μM ddATP; 643 μM dATP; 643 μM dCTP; 643 μM dGTP; 643 μM 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.  
         [0096]    C-Mix: 2.14 μM ddCTP; 643 μM dATP; 643 μM dCTP; 643 μM dGTP; 643 μM 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.  
         [0097]    G-Mix: 2.14 μM ddGTP; 643 μM dATP; 643 μM dCTP; 643 μM dGTP; 643 μM dTTP; 330 nM total of forward primers and 330 nM total of reverse primers; 1 mM Tris-HCl, pH 8.0 at 5° C., 0.1 mM EDTA.  
         [0098]    T-Mix: 2.14 μM ddTTP; 643 μM dATP; 643 μM dCTP; 643 μM dGTP; 643 μM dTTP; 330 nM total of forward primers and 330 nM total of reverse primers  
         [0099]    Both primers are labeled, for example with Cy5.0 and Cy5.5, respectively.  
         [0100]    Second Reverse Transcriptase Region  
         [0101]    A-Mix: 1.07 μM ddATP; 643 μM dATP; 643 μM dCTP; 643 μM dGTP; 643 μM dTTP; 330 nM total of forward primers and 330 nM total of reverse primers  
         [0102]    C-Mix: 2.14 μM ddCTP; 643 μM dATP; 643 μM dCTP; 643 μM dGTP; 643 μM dTTP; 330 nM total of forward primers and 330 nM total of reverse primers  
         [0103]    G-Mix: 2.14 μM dGTP; 643 μM dATP; 643 μM dCTP; 643 μM dGTP; 643 μM dTTP; 330 nM total of forward primers and 330 nM total of reverse primers  
         [0104]    T-Mix: 2.14 μM ddTTP; 643 μM dATP; 643 μM dCTP; 643 μM dGTP; 643 μM 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.  
         [0105]    P2 Protease Region  
         [0106]    A-Mix: 1.07 μM ddATP; 643 μM dATP; 643 μM dCTP; 643 μM dGTP; 643 μM dTTP; 330 nM total of forward primers and 330 nM total of reverse primers  
         [0107]    C-Mix: 2.14 μM ddCTP; 643 μM dATP; 643 μM dCTP; 643 μM dGTP; 643 μM dTTP; 330 nM total of forward primers and 330 nM total of reverse primers  
         [0108]    G-Mix: 2.14 μM ddGTP; 643 μM dATP; 643 μM dCTP; 643 μM dGTP; 643 μM dTTP; 330 nM total of forward primers and 330 nM total of reverse primers  
         [0109]    T-Mix: 2.14 μM ddTTP; 643 μM dATP; 643 μM dCTP; 643 μM dGTP; 643 μM dTTP; 330 nM total of forward primers and 330 nM total of reverse primers  
         [0110]    Both primers are labeled, for example with Cy5.0 and Cy5.5, respectively.