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WO1992016180A2 - Detection de la resistance a la 3'-azido-3'-desoxythymidine - Google Patents

Detection de la resistance a la 3'-azido-3'-desoxythymidine Download PDF

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Publication number
WO1992016180A2
WO1992016180A2 PCT/US1992/002037 US9202037W WO9216180A2 WO 1992016180 A2 WO1992016180 A2 WO 1992016180A2 US 9202037 W US9202037 W US 9202037W WO 9216180 A2 WO9216180 A2 WO 9216180A2
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Prior art keywords
nucleotide
sequence
seq
unknown
type
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Application number
PCT/US1992/002037
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English (en)
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WO1992016180A3 (fr
Inventor
Thomas Raymond Gingeras
Kevin J. Barringer
Douglas D. Richman
Patricia C. Prodanovich
Geneva Ruth Davis
Original Assignee
Siska Diagnostics, Inc.
The Regents Of The University Of California
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by Siska Diagnostics, Inc., The Regents Of The University Of California filed Critical Siska Diagnostics, Inc.
Publication of WO1992016180A2 publication Critical patent/WO1992016180A2/fr
Publication of WO1992016180A3 publication Critical patent/WO1992016180A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Definitions

  • HIV-1 Human Immunodeficiency Virus
  • AIDS Acquired Immune Deficiency Syndrome
  • ARC AIDS-related complex
  • the virus enters these T-cells via a specific receptor integral to the CD4 T-cell specific antigen. Upon entry, the virus directs synthesis of reverse transcriptase from the pol gene, thereby enabling the RNA virus to generate a duplex DNA copy capable of integrating the host cell genome, from which further virus production is directed.
  • the HIV-1 virus leaves the host individual open to a variety of
  • EP 0 378 643 discloses a therapeutic composition comprising a soluble T4 protein and either azidothymidine or a glucoside inhibitor.
  • U.S. Patent Nos. 4,866,036 and 4,866,035 disclose use of the combination of dipeptidyl glucofuranose compounds and an antiviral agent such as anidothymidine, ansamycin, ribavirin, dioxycytidine, or foscarnet.
  • antiviral compound azidothymidine (AZT)
  • AZT an analog of thymidine
  • AZT administered to AIDS patients produces an increase in CD4 positive cells and usually a decrease in detectable serum levels of the viral p24 protein, although these
  • a major long-term drawback to AZT therapy is a tendency for resistance to the drug to develop. This is found to occur by degrees over a period of prolonged use, and involves certain mutations in the pol gene,
  • resistance may have predictive value in planning
  • an assay for detecting the genotype of a mutant or wild type marker, which are distinguished by one or more base changes in the chromosomal DNA at one or more loci resulting in an amino acid substitution in the protein encoded by the gene in question.
  • a nucleic acid fraction is extracted from cells expected to express the mutant or wild-type marker.
  • the nucleic acid fraction is then amplified by nucleic acid amplification techniques such as polymerase chain reaction (PCR) or self-sustained sequence replication (3SR).
  • Primers for such amplification are selected which span the target marker region.
  • the amplified nucleic acids are then separated by capturing same on a capture means, such as a homologous sequence having a sequence distinct from the marker sequence affixed to a support means, and washing away the non-homologous nucleic acids.
  • a probe is constructed which hybridizes either to the mutant or wild-type marker regions.
  • These probes have reporter means such as a radioactive substance or fluorophor to permit detecting the hybridization of the probes to the captured
  • the assay target is a sequence of the HIV-1 pol gene which contains mutations or the corresponding wild-type alleles at the 67, 70, 215 and 219 amino acid positions.
  • the assay is performed by first extracting the nucleic acid fraction from cells obtained from patients with a varying degree of AZT resistance, amplifying a nucleic acid sequence in the nucleic acid fraction spanning the AZT resistant
  • mutant-containing region from about nucleotide 2330 to
  • the probe sequences which are necessary for the differential determination of the correct AZT resistance genotype comprise a first family of probe sequences for detecting in solution hybridization assays the mutant or wild-type genotype of AZT resistance in the HIV-1 pol gene comprising a first family of probe sequences having a nucleotide sequence of 24 to 30 nucleotides
  • nucleotide sequence containing an inosine nucleotide at position 2330 and a guanidine nucleotide at position 2340, said sequence extending from the inosine positioned substantially 2-4 nucleotides from the 5'-terminus to a nucleotide located 3' of the said guanidine nucleotide such that said guanidine nucleotide is located
  • nucleotide sequence containing an adenosine nucleotide at position 2330 and an inosine nucleotide at position 2340 comprising a nucleotide sequence containing an adenosine nucleotide at position 2330 and an inosine nucleotide at position 2340, the sequence extending from a nucleotide located substantially 12 to 15 nucleotides 5* of said adenosine nucleotide to the inosine nucleotide located 3-6 nucleotides from the 3'-terminus, a probe specific for the wild-type genotype comprising a nucleotide sequence containing a guanidine at position 2330 and an adenosine nucleotide at position 2340, said sequence extending from said inosine positioned substantially 3-7 nucleotides from the 5*-terminus to a nucleotide located 3' of said adenosine nucleotide such that said a
  • nucleotide 2787 such that said nucleotide 2787 is located spacedly at substantially the midpoint in the sequence, a probe specific for the mutant allele at position 2787 comprising a nucleotide sequence containing a cytosine nucleotide at position 2787, said sequence being
  • nucleotide at position 2776 and a adenosine nucleotide at position 2787, said sequence being substantially symmetrical about the midpoint nucleotide between
  • nucleotides 2774 and 2787 are nucleotides 2774 and 2787.
  • the present invention provides an assay for detecting the genotype of a mutant or wild-type marker utilizing a nucleic acid probe-based system.
  • the model assay detects the genotype of AZT resistance which results from point mutations at 4 known sites.
  • the assay method is also applicable to other drug
  • nucleic acid fraction from a tissue source must be extracted. Most commonly, a good nucleic acid preparation can be obtained utilizing the phenol/chloroform extraction method described by Maniatas, et al. Molecular Cloning: A Laboratory Manual, 1982. A method of nucleic acid
  • PCR polymerase chain reaction
  • 3SR self-sustaining sequence replication
  • the amplified sequences are first captured on solid support means by a capture means comprising a nucleic acid sequence
  • the support means may be any solid material known in the assay art to effect separations of target sequence from nonspecific
  • beads 1-200 microns in diameter made of polyacrylamide or polystyrene.
  • primer sequences for amplifying the region containing the mutational positions conferring AZT resistance are set forth in Table 1.
  • primers utilized in 3SR require at one member of the primer pair to contain a T7 RNA polymerase promoter sequence.
  • probe sequences of the present invention which detect mutations, either together or singly, at the amino acid 67 and 70 positions are given in Table 2.
  • Preferred probe sequences which differentially detect mutations, either together or singly, at the amino acid 215 and 219 positions are set forth in Table 3.
  • the third to the left column of each of the probe tables gives the genotype of the 67/70 region for each of the mutant and wild-type possibilities.
  • column 3 of Table 3 provides the genotype of 215/219 region for each such mutant and wild-type possibility.
  • Class 1 probes are oligonucleotides that monitor both loci of a specific region
  • Class 2A oligonucleotides include shorter probes whose sequences encompass only one of the amino acid codons implicated in AZT resistance.
  • Class 2B probes are similar to class 1 probes, but contain a nucleotide analog, inosine, at one of the two mutant positions. The use of inosine, which partially base pairs equally well with all other nucleotides, neutralizes mismatches and thus enables these probes to be used for one specific locus. For example, a probe whose genotype at the 67/70 region is +/- hybridizes specifically to a +/- target under stringent conditions.
  • Oligonucleotides have been synthesized which specifically detect each mutant sequence. Additionally, an inosine residue at the non-AZT associated degeneracy at the first base of the codon for the amino acid 214 has been
  • Class 2C probes span both loci with one locus centered and the other near the end of the probe. This asymmetric alignment of mutant sites would be predicted to enable the probe to function in a manner analogous to the inosine-containing probes described above. This prediction is based upon the assumption that a mismatch toward either end of a probe would have little effect upon the stability of the duplex. Therefore, probes of this structure would be specific for the locus which is centered in the sequence. Class 2D are similar to 2C probes but contain inosine at the mutation sites located near the ends of the probes, thus further minimizing the end-of-probe mismatches. The probe construction strategy is further illustrated in figure 1, which gives the characteristics of the various probe classes.
  • Plasmids containing one of the four mutations implicated in the generation of resistance to AZT were constructed by site-directed mutagenesis method similar to that reported by Zollar, et. al. Methods in
  • oligonucleotide containing the desired mutation were annealed to 1 pmol of a single strand M13mp18 clone containing an approximately 1700 nt Asp718-BglII insert from pARV.
  • This fragment contains the portion of the pol gene spanning the amino acid 67, 70, 215 and 219.
  • the primers were extended using the Klenow fragment of E.
  • nitrocellulose filters (Millipore, HATF). Phage DNA on the filters were denatured, hybridized with 32P-labeled oligonucleotide probes specific for the desired mutations and exposed to film. Phage mini-preps were prepared from several positive plaques and these were used in a second round of screening as described in Maniatas, et. al..
  • the plasmid RTMC/3 (gift from B. Larder) contained all four point mutations. A 2.6 kb
  • RNA transcripts were made from these plasmids using 1 to 10 ug of linearized plasmid and conditions described by the manufacturer.
  • 3SR reactions were carried out as described previously in Guatolli, et. al., PNAS, 87:1874 (1990) except that 10 mM KCl was used instead of 20 mM NACl, BSA was omitted and the incubations were carried out at 42oC. instead of 37oC. Only the antisense primer
  • oligonucleotide contains a T7 RNA polymerase promoter sequence.
  • 3SR reactions were denatured at 100'C. for 5 minutes, annealed at 42oC for 2 minutes and 10 U of reverse transcriptase were added. Reactions were incubated at 42oC. for 10 minutes, the denaturation/annealing steps repeated, then 3SR reaction initiated.
  • BBSH Differential Bead-based Sandwich Hybridization
  • the detection of 3SR amplification products and the determination of the genotype of the 3SR RNA was accomplished by use of a differential BBSH procedure. Similar to the BBSH protocol described previously
  • differential BBSH assays used 2 ml microcolumns (Isolab, Inc.) which contain 25 mg of
  • Trisacryl Oligobeadstm (10). To these beads, 100 femtomoles of 32P-labeled detection oligonucleotide, and 10-2 to 10-3 aliquots of the 3SR product in a total volume of 30 ul were added. These hybridization
  • probes for the 215/219 region contain an inosine residue at the first nucleotide at the amino acid 214 codon to compensate for a sequence heterogeneity between several HIV-1 isolates. Additionally, probes that contain a mutant 215 locus are degenerate at the second nucleotide of the amino acid 215 codon. This position has been shown to have two possible amino acid substitutions occur.
  • the assay to determine the genotype of the 3SR amplified material is a thermal melt/batch elution assay.
  • the 3SR amplification products from each mutant region was analyzed by four specific detection probes
  • each amplification reaction was first subjected to low stringency analyses consisting of a 42oC hybridization step followed by 2 ⁇ SSC (where 1 ⁇ equals 0.15 M NaCl, 15 MM sodium citrate) was at 42oC. This was followed by a second high stringency
  • differential hybridization For the amino acid 67/70 region, the differential BBSH reactions are hybridized at 42oC, then washed at 50oC with 0.75 to 1.0 ⁇ SSC washes (see Table 6A). The amino acid 215/219 containing BBSH reactions are hybridized at 55oC followed by washes with 0.5 ⁇ SSC at 55oC
  • oligonucleotide detection probe was designed to monitor two of the specified codons simultaneously.
  • 215-219 region contained inosine at the first base of codon 214 and probes 90-304 and 90-302 were composed of mixed population probes to account for the possibility of either mutation at position 215.
  • the phenylalamine (pPol 215P-218) and tyrosine (pPol 215T-18) mutant codons present at the 215 position were detected with almost equal efficiency.
  • Differential elution can be accomplished by block elution at a specific high or low stringency salt wash. Alternatively, by introducing a gradient of decreasing salt concentration, elution of the detection probe at an optimal stringency can be detected.
  • the protocol for sequential elution (block) is illustrated in Figure 3.
  • HIV-1 viruses isolated from the PBMCs of seven patients who had received AZT therapy for 5 to 26 months were analyzed first by the separate 3SR
  • Sample 1429-1 also exhibited a requirement for lower wash stringency in the BBSH assay to determine the genotype of the HIV-1 pol gene in this sample.
  • the sequence of the 67-70 region of the pol gene of this virus showed no sequence differences in the region covered by the detection probe. However, sequence differences were observed in the region surveyed by the oligonucleotide used to capture the target on the bead support. These sequence variations appear to have led to a lower hybridization efficiency of the complementary 90-36 (wt/wt) probe.
  • 89-441 hybridized 40 bases away from the 67 and 70 codons and provided a measure of the amounts of 3SR product in each of the differential BBSH assays.
  • the samples from seven patients analyzed by the 3SR/differential BBSH assay were also analyzed using PCR/differential Souther hybridization method.
  • Viral isolates were obtained directly from the PBMCs and viral pools.
  • Table 6 correlates the results obtained by 1) each set of amplification-mediated hybridization methods (3SR and PCR), 2) the nucleotide sequence analyses of the 3SR products and 3) the AZT-susceptibility assays of the viral isolates obtained from each sample.
  • genotypic results obtained by the 3SR/differential BBSH and nucleotide sequence analyses are in agreement for all codons except for the 67 and 70 codons of samples 1381-4 and J821-1.
  • the 3SR/differential BBSH assay detects the predominant mutant forms of the mixed virus population detected by the sequence analyses.
  • 3SR/differential BBSH and PCR/Souther hybridization analyses for the viral isolates are substantially in agreement except for samples G685-2 and 1312-6.
  • the mixed population of viruses detected by the PCR/Southern hybridization assay for sample J821-1 is composed of predominantly mutant virus which agrees with both the 3SR/differential BBSH and sequence analyses.
  • sample G685-6 may be attributable to the fact two different passages of this viral isolate were analyzed by the PCR/differential Southern hybridization and 3SR/differential BBSH assays. Such variation between viral passages suggest similar discrepancies should be observed between PBMC and
  • samples G685-6 and J821-1 possess the same genotype (wt/mut/mut/wt) at each of the four
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  • Chemical & Material Sciences (AREA)
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  • Engineering & Computer Science (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
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Abstract

Méthode de détection du génotype des allèles mutants et de phénotype sauvage au niveau d'une pluralité d'emplacements associés à la résistance au médicament AZT. La méthode consiste à utiliser une série de sondes aptes à s'hybrider à une région amplifiée du gène pol du VIH-1. La structure de méthode consistant à amplifier un acide nucléique rare puis à le capturer sur une phase solide, ainsi qu'à détecter les séquences capturées à l'aide d'une sonde marquée, s'applique aux méthodes déterminant la résistance à d'autres médicaments et impliquant des mutations ponctuelles.
PCT/US1992/002037 1991-03-13 1992-03-12 Detection de la resistance a la 3'-azido-3'-desoxythymidine WO1992016180A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US66854991A 1991-03-13 1991-03-13
US668,549 1991-03-13
US75414691A 1991-09-03 1991-09-03
US754,146 1991-09-03

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WO1992016180A2 true WO1992016180A2 (fr) 1992-10-01
WO1992016180A3 WO1992016180A3 (fr) 1992-10-29

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996002557A1 (fr) * 1994-07-19 1996-02-01 Gen-Probe Incorporated Composes et procedes permettant d'inhiber la propagation du virus de l'immunodeficience humaine
EP0727497A1 (fr) * 1995-02-17 1996-08-21 F. Hoffmann-La Roche Ag Amorces et sondes pour détection de VIH
WO1997027332A1 (fr) * 1996-01-26 1997-07-31 Innogenetics N.V. Procede de detection de mutations induites par des medicaments dans le gene de la transcriptase reverse
US5962665A (en) * 1997-06-16 1999-10-05 Abbott Laboratories Nucleic acid primers and probes for detecting HIV-1 and HIV-2
EP1017856A1 (fr) * 1997-09-26 2000-07-12 Visible Genetics Inc. Procede et ensemble de materiel d'evaluation de mutations vih
US6265152B1 (en) * 1995-12-22 2001-07-24 Visible Genetics Inc. Method and kit for evaluation of HIV mutations
WO2003018835A3 (fr) * 2001-08-23 2004-03-25 Hvidovre Hospital Procede de detection rapide d'haplotypes
US6830887B2 (en) 1997-03-18 2004-12-14 Bayer Healthcare Llc Method and kit for quantitation and nucleic acid sequencing of nucleic acid analytes in a sample
EP2035578A2 (fr) * 2006-06-09 2009-03-18 Conexio 4 Pty Ltd Identification d'une molécule d'acide nucléique
US7666600B2 (en) * 2003-12-19 2010-02-23 Gen-Probe Incorporated Cross-reactive primers for amplifying the nucleic acids of HIV-1 and HIV-2

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8918226D0 (en) * 1989-08-09 1989-09-20 Wellcome Found Dna assays
WO1991010746A1 (fr) * 1990-01-10 1991-07-25 Chiron Corporation Transcriptions d'arn-polymerase dependante d'adn servant de molecules reportees pour l'amplification de signaux dans les analyses d'hybridation d'acide nucleique
IL97222A (en) * 1990-02-16 1995-08-31 Orion Yhtymae Oy Method and reagent for determining specific nucleotide variations

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0697414A1 (fr) * 1994-07-19 1996-02-21 Gen-Probe Incorporated Composés et méthodes pour inhiber la propagation de VIH
US5733781A (en) * 1994-07-19 1998-03-31 Gen-Probe Incorporated Oligonucleotides and methods for inhibiting propagation of human immunodeficiency virus
WO1996002557A1 (fr) * 1994-07-19 1996-02-01 Gen-Probe Incorporated Composes et procedes permettant d'inhiber la propagation du virus de l'immunodeficience humaine
EP0727497A1 (fr) * 1995-02-17 1996-08-21 F. Hoffmann-La Roche Ag Amorces et sondes pour détection de VIH
US5599662A (en) * 1995-02-17 1997-02-04 Hoffmann-La Roche Inc. Oliconucleotide primers and probes for the detection of HIV-1
US6265152B1 (en) * 1995-12-22 2001-07-24 Visible Genetics Inc. Method and kit for evaluation of HIV mutations
WO1997027332A1 (fr) * 1996-01-26 1997-07-31 Innogenetics N.V. Procede de detection de mutations induites par des medicaments dans le gene de la transcriptase reverse
AU719691B2 (en) * 1996-01-26 2000-05-18 Innogenetics N.V. Method for detection of drug-induced mutations in the reverse transcriptase gene
US6087093A (en) * 1996-01-26 2000-07-11 Innogenetics N.V. Method for detection of drug-induced mutations in the reverse transcriptase gene
US6713251B2 (en) 1996-01-26 2004-03-30 Innogenetics N.V. Method for detection of drug-induced mutations in the reverse transcriptase gene
US6331389B1 (en) 1996-01-26 2001-12-18 Innogenetics N.V. Method for detection of drug-induced mutations in the reverse transcriptase gene
US6830887B2 (en) 1997-03-18 2004-12-14 Bayer Healthcare Llc Method and kit for quantitation and nucleic acid sequencing of nucleic acid analytes in a sample
US6232455B1 (en) * 1997-06-16 2001-05-15 Abbott Laboratories Nucleic acid primers and probes for detecting HIV-1 and HIV-2
US5962665A (en) * 1997-06-16 1999-10-05 Abbott Laboratories Nucleic acid primers and probes for detecting HIV-1 and HIV-2
EP1017856A1 (fr) * 1997-09-26 2000-07-12 Visible Genetics Inc. Procede et ensemble de materiel d'evaluation de mutations vih
WO2003018835A3 (fr) * 2001-08-23 2004-03-25 Hvidovre Hospital Procede de detection rapide d'haplotypes
US7666600B2 (en) * 2003-12-19 2010-02-23 Gen-Probe Incorporated Cross-reactive primers for amplifying the nucleic acids of HIV-1 and HIV-2
EP2251442A1 (fr) * 2003-12-19 2010-11-17 Gen-Probe Incorporated Compositions, méthodes et kits destinés à la détection des acides nucléiques du VIH-1 et du VIH-2
US8318432B2 (en) 2003-12-19 2012-11-27 Gen-Probe Incorporated Cross-reactive hybridization probe for detecting HIV-1 and HIV-2 nucleic acids in the P31 gene sequence
EP2035578A2 (fr) * 2006-06-09 2009-03-18 Conexio 4 Pty Ltd Identification d'une molécule d'acide nucléique
EP2035578A4 (fr) * 2006-06-09 2010-01-27 Conexio 4 Pty Ltd Identification d'une molécule d'acide nucléique

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AU1741692A (en) 1992-10-21

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