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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
  • C12Q1/68—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
  • C12Q1/6851—Quantitative amplification

Definitions

• This invention relates to diagnostic assays for nucleic acids that include amplification by the polymerase chain reaction (PCR).
• PCR polymerase chain reaction
• the solid matrices may be of a variety of materials such as agarose, polyacrylaminde, nylon, gelatin, alginate, carrageenan, cellulose, silica gel, titanium sponge, cross-linked agarose, dextran or polyethylene glycol.
• the amplification system may be any system for exponential amplification of nucleic acids in vitro, such as viral RNA-directed RNA polymerases, PCR or isothermal multienzyme (3SR) amplification, and may be introduced into a gel matrix either prior to gel formation or, particularly when the conditions of gel preparation are too strong for labile amplification reagents, by impregnation of pre-cast gels.
• An aspect of this invention is a type of multiplex clinical assay for relatively rare nucleic acid targets residing in samples (for example, samples of blood from a human patient) containing other target nucleic acids and non-target nucleic acids, which are present in manifold excess compared to the relatively rare target.
• the diagnostic assay utilizes the polymerase chain reaction (PCR) process. If the target nucleic acid is RNA, reverse transcriptase-PCR (RT-PCR) is used, of course.
• nucleic acids (DNA, RNA) isolated from a sample need to be diluted in the solid matrix, by spreading, only sufficiently to permit the growth of individual colonies from the relatively rare target molecules.
• Nucleic acids from the sample need not be diluted sufficiently that other, more abundant nucleic acid targets would form identifiable individual colonies in a multiplex assay of both targets (or all targets, if more than two are amplified). For example, if a sample contains nucleic acids from two viruses, one can amplify nucleic acid target from the rarer of the two to form identifiable individual colonies even though the more abundant of the two is present in such a> large concentration, ten or more times as much, that even without amplification it appears on probing to permeate the matrix entirely.
• each target is amplified by a pair of primers that, false priming aside, is specific to that target relative to other nucleic acids in the sample.
• two different viral targets one rare and one abundant are amplified using a pair of primers specific to each, and colonies of each are identified utilizing probes specific to each.
• a rare mutant allele can be detected in a sample containing abundant wild-type allele by using a primer pair that is specific for the mutant relative to the wild type.
• a primer pair that is specific for the mutant relative to the wild type.
• mutants differing from their corresponding wild types by a single nucleotide one could utilize a primer whose 3' terminal nucleotide is complementary to the mutant at that single nucleotide.
• a high specificity primer such as is disclosed in published PCT patent application WO 00/71562 Al .
• Additional targets, if present, having concentrations intermediate the rare target and the abundant will produce countable quantities at intermediate dilutions, such as occur with a dilution series.
• the multiplex assays of this invention utilize polyacrylamide gel in a thin layer as the solid matrix. Further, the polyacrylamide gel is pre-cast, washed and dried as a plain gel, and later impregnated with PCR reagents prior to use, which we have found to impart improved reproducibility, important for diagnostic assays, as compared to mixing the amplification reagents with gel-producing ingredients prior to casting.
• Preferred embodiments of assays according to this invention also utilize improved nucleic-acid isolation from the sample, if a blood sample, that includes washing pellets of isolated nucleic acids with a saline-alcohol solution, which we have discovered removes substances that are inhibitory to PCR and reverse transcription.
• nucleic acids extracted from a specimen are introduced into a thin polyacrylamide gel, together with, for example, Thermus thermophilus (Tth)DNA polymerase (which can function both as a DNA polymerase and a reverse transcriptase in the presence of Mn 2+ ions) 2 ' 3 , 2'-deoxyribonucleoside 5'-triphosphate (dNTP) substrates, and virus-specific oligonucleotide primers.
• the gel is incubated under conditions appropriate for reverse transcription (RT), followed by polymerase chain reaction (PCR) 4 temperature cycles. During incubation, target molecules (in this embodiment, viral DNA or RNA)produce DNA colonies at discrete locations within the gel.
• RT reverse transcription
• PCR polymerase chain reaction
• the identity of DNA colonies is revealed by hybridization with virus- specific labeled probes.
• the number of DNA colonies hybridizable with a particular probe indicates the copy number of the respective viral DNA or RNA in the analyzed sample.
• S-PCR conventional solution PCR
• the new method shows better sensitivity and reliability, eliminates the interference between assayed targets and from the background DNA synthesis caused by primer-dimers or by mispriming on patient's own nucleic acids, and provides for a direct determination of target titer.
• MCT is a method of amplifying nucleic acids (RNA or DNA)in a matrix, which may be a gel.
• RNA or DNA nucleic acids
• a matrix which may be a gel.
• the progeny of each molecule forms a colony, rather than spreads throughout the reaction volume.
• Each colony comprises many copies of one original molecule (i. e., a clone), and the number of colonies indicates the number of nucleic acid molecules initially present in the gel.
• the unique feature of MCT distinguishing it from other methods for nucleic acid amplification is that amplified molecules are spatially separated. This results in weakening or (given the sample is properly diluted) complete elimination of the competition between molecular species, and allows individual amplifiable molecules to be monitored, counted and analyzed.
• the gel was agarose containing
• Q ⁇ replicase (the RNA-directed RNA polymerase of bacteriophage Q ⁇ ) 7 and ribonucleoside 5'-triphosphates.
• Q ⁇ -MCT provides for growing the colonies of RQ RNAs (i. e., RNAs Replicable by Q ⁇ replicase, including the natural Q ⁇ phage satellites ), and proved to be a powerful tool for the in vitro studies of very rare events of RNA recombination 8 ' 9 . In principle, it is possible to use Q ⁇ -MCT for diagnostic purposes, e.
• RNA fragments are intrinsically recombinogenic and can produce RQ RNAs in the absence of a target and ligase 8 ' 9 , and many foreign inserts, even short, do inhibit replication of RQ RNAs due to the structural requirements that Q ⁇ replicase imposes on its templates 7 ' 11 .
• PCR-MCT in which DNA colonies from two or more targets are grown by carrying out PCR in heat-resistant polyacrylamide gel.
• virtually any DNA can be amplified in PCR, which is now widely used in basic research, biotechnology and clinical diagnostics .
• our original PCR-MCT protocol has been reproduced by others and, inasmuch as the equipment and reagents that are required for carrying out PCR are readily available on the market, we believe that PCR-MCT can become a routine laboratory technique.
• RNA recovery to that high level as a result of the attempts to avoid ribonuclease contaminations (see Examples), we believe that it can be further increased by additionally purifying the protein preparations used in the assay, Tth DNA polymerase and bovine serum albumin (BSA).
• BSA bovine serum albumin
• target-specific e.g., virus-specific
• oligonucleotides anneal (despite some mismatches)and prime on human sequences resulting in a background DNA amplification.
• the background amplification can be monitored by gel electrophoresis of the reaction products, and it interferes with the amplification of a target in S-PCR assays (Fig. 3a).
• the multiplex MCT method according to this invention is able to overcome the conceptual problems inherent to S-PCR, such as the sensitivity loss due to a competition from background amplification (caused by false priming on non-target sequences often present in the sample in a great excess over the target), preferential amplification of some templates over others, and template recombination (during the amplification of heterozygous samples and multigene families, and in multiplex PCR). 14 In MCT, different molecules of the same target amplify at different locations, which eliminates any competition and prevents recombination between them. In multiplex assays according to this invention, we have discovered that this holds true even when another target is present in high amount and not spread sufficiently to produce identifiable colonies.
• MCT quantitative assays
• S-PCR measures it indirectly, by referring to the signal produced by a known number of molecules of an internal standard (IS) added to the sample; therefore, the result depends on whether the target and IS are equally efficient templates.
• IS internal standard
• reliable measurements can only be done if the quantities of the two templates differ by ⁇ 1.5- fold; therefore, careful multiple-reaction calibrations need to be carry out to assess a single target species in one sample.
• HBV DNA target we used plasmid obtained by ligating, into pTZ19R BarnHI-fragment encoding core antigen HbcAg, the HBV subtype ayw genome 16 that had been excised from pUHBc (ref. 17).
• RNA target As an HIV-I RNA target, we used a 879 nt-long run-off transcript from a Sm ⁇ L-digestsd plasmid 10 carrying, downstream from T7 promoter, fragment 4230-5091 of HIV-I strain NL4-3; the transcript has been treated with RQl Rnase-free DNase (Promega, Madison, WI) to eliminate the plasmid DNA; the resulting preparation produced no specific PCR product if RT step was omitted.
• Q ⁇ RNA was isolated by phenol extraction from wild- type Q ⁇ phage that was purified as described 5 .
• GCTTAATACGACTCACTATAGGGCTA- CTGTGGAGTTACTCTCGTTTTTGC-3 ' and 5'-GTCTATAAGCTGGAGGAGTGCGA- ATC-3' matching positions 1933-1 960 and 2277-2302 of the HBV genome, respectively
• 5'- CAAGTAGACTGTAGTCCAGGAATAT-3' and 5'- GCTTAATACGACTCACTATAG- GGGATTGTAGGGAATGCCA-3' matching positions 4386-4410 and 4646-4665 of the HIV genome, respectively
• CAATGCTCAGGAGACTCTAAGGCTTCCCGATACAGAGCTG-3' positions 2006-2045 of HBV genome
• 5'-CAGTACATACAGACAATGGC- AGCAATTTCACCAG-3' positions 4564-4597 of HIV genome
• DNA polymerases The coding region of polA gene (GenBank accession No. D28878) was PCR-amplifted, starting with chromosomal T. thermophilus HB8 DNA, using primers that introduced upstream site Ndel and His 6 -coding sequence and downstream site EcoRV, cloned in pETl lc between sites Ndel and BamHl and expressed in B834(DE3)/pLysS Escherichia coli cells 18 . Highly purified N-His 6 -tagged Tth DNA polymerase was isolated from these cells by heating lysate and (NH ) 2 SO 4 precipitation , followed by chromatography on Zn -iminodiacetate-Sepharose .
• N- His 6 -tagged Taq DNA polymerase was obtained similarly. Isolation of nucleic acids from total blood. We used anonymous citrate-treated blood samples obtained from healthy donors and tested HBV and HIV-1 negative at a blood transfusion station; IRB required no informed consent in this case.
• Residual ribonucleases were inactivated with iodoacetamide 22 at pH 8.0.
• the yield of total nucleic acids (DNA +RNA) was 30- 60 ⁇ g per ml of blood, as determined spectrophotometrically.
• PCR cycles were performed as follows: the first 3 cycles [melting (94 °C, 15 s), annealing (4 s) and extension (72 °C, 60 s)] were followed by 40 cycles with the melting time being reduced to 6 s, followed by incubation at 72 °C for 5 min. Annealing temperature was 50 °C for HIV-1 target or 60 °C for Q ⁇ target, either for HBV target.
• RNA hybridization probes were prepared by T7 RNA polymerase transcription in the presence of [ ⁇ - 32 P]ATP (ref.
• oligonucleotides were labeled using [ ⁇ - PjATPand polynucleotide kinase (Roche Molecular Biochemicals)according to the manufacturer 's protocol.
• RNA replication RNA catalytic for single-strand release. Virology 184, 595-608 (1991 ). 12. Taylor, G. R., and Robinson, P. The polymerase chain reaction: from functional genomics to high school practical classes. Curr. Opin. Biotechnol. 9,35-42 (1998).

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  • 2002-06-21 AU AU2002330802A patent/AU2002330802A1/en not_active Abandoned
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