WO1997029210A3 - Fluorescence detection assay for homogeneous pcr hybridization systems - Google Patents

Fluorescence detection assay for homogeneous pcr hybridization systems Download PDF

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Publication number
WO1997029210A3
WO1997029210A3 PCT/US1997/001755 US9701755W WO9729210A3 WO 1997029210 A3 WO1997029210 A3 WO 1997029210A3 US 9701755 W US9701755 W US 9701755W WO 9729210 A3 WO9729210 A3 WO 9729210A3
Authority
WO
WIPO (PCT)
Prior art keywords
oligonucleotide
nucleic acid
pcr
probe
acid sequence
Prior art date
Application number
PCT/US1997/001755
Other languages
French (fr)
Other versions
WO1997029210A2 (en
Inventor
Cesare Joseph L Di
Original Assignee
Perkin Elmer Corp
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.)
Filing date
Publication date
Application filed by Perkin Elmer Corp filed Critical Perkin Elmer Corp
Priority to JP52862197A priority Critical patent/JP3415627B2/en
Priority to DE69702649T priority patent/DE69702649T2/en
Priority to AT97904214T priority patent/ATE195004T1/en
Priority to AU18566/97A priority patent/AU700521B2/en
Priority to EP97904214A priority patent/EP0870063B1/en
Priority to CA002243759A priority patent/CA2243759C/en
Publication of WO1997029210A2 publication Critical patent/WO1997029210A2/en
Publication of WO1997029210A3 publication Critical patent/WO1997029210A3/en

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]

Abstract

An improved assay system for homogeneous assay detection and/or measuring target nucleic acid sequence replicated in a PCR amplification procedure is provided by employing a nucleic acid polymerase having 5' to 3' exonuclease activity and devoid of 3' to 5' exonuclease activity, oligonucleotide analytical probe blocked against chain extension at its 3' terminus and labeled at its 5' terminus with an energy transfer donor fluorophore, and oligonucleotide detection probe labeled at its 3' terminus with an energy transfer acceptor fluorophore, said probes being complements of each other but differing in nucleotide length so that their Tm's are at least 10 °C apart, the Tm of the oligonucleotide analytical probe is equal to or greater than the Tm's of the oligonucleotide primers used in the PCR amplification procedure and the Tm of the oligonucleotide detection probe is lower than the temperature required to conduct the PCR thermal cycling steps of the PCR amplification procedure so that during the PCR thermal cyclic steps 5' fluorophore labeled nucleotide fragments are produced during the PCR extension phase by the 5' to 3' exonuclease activity of the nucleic acid polymerase on oligonucleotide analytical probe annealed to denatured strands of the target nucleic acid sequence. Detection of measurement of either (a) the 5' fluorophore labeled nucleotide fragments produced during the thermal cyclic steps and measured by fluorescence polarization or (b) oligonucleotide analytical probe hybridized to oligonucleotide detection probe measured spectrophotometrically by energy transfer measurement, provide a measure of the amount of oligonucleotide analytical probe used up in the amplification of the target nucleic acid sequence and thus provide a measure of amount of target nucleic acid sequence amplified in the PCR replication procedure.
PCT/US1997/001755 1996-02-05 1997-01-31 Fluorescence detection assay for homogeneous pcr hybridization systems WO1997029210A2 (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
JP52862197A JP3415627B2 (en) 1996-02-05 1997-01-31 Fluorescence detection assays for homogeneous PCR hybridization systems
DE69702649T DE69702649T2 (en) 1996-02-05 1997-01-31 FLUORESCENCE DETECTION TEST FOR HOMOGENEOUS PCR HYBRIDIZATION SYSTEMS
AT97904214T ATE195004T1 (en) 1996-02-05 1997-01-31 FLUORESCENCE DETECTION TEST FOR HOMOGENEOUS PCR HYBRIDIZATION SYSTEMS
AU18566/97A AU700521B2 (en) 1996-02-05 1997-01-31 Fluorescence detection assay for homogeneous PCR hybridization systems
EP97904214A EP0870063B1 (en) 1996-02-05 1997-01-31 Fluorescence detection assay for homogeneous pcr hybridization systems
CA002243759A CA2243759C (en) 1996-02-05 1997-01-31 Fluorescence detection assay for homogeneous pcr hybridization systems

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US08/596,560 US5716784A (en) 1996-02-05 1996-02-05 Fluorescence detection assay for homogeneous PCR hybridization systems
US08/596,560 1996-02-05

Publications (2)

Publication Number Publication Date
WO1997029210A2 WO1997029210A2 (en) 1997-08-14
WO1997029210A3 true WO1997029210A3 (en) 1997-10-09

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Family Applications (1)

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PCT/US1997/001755 WO1997029210A2 (en) 1996-02-05 1997-01-31 Fluorescence detection assay for homogeneous pcr hybridization systems

Country Status (8)

Country Link
US (1) US5716784A (en)
EP (1) EP0870063B1 (en)
JP (1) JP3415627B2 (en)
AT (1) ATE195004T1 (en)
AU (1) AU700521B2 (en)
CA (1) CA2243759C (en)
DE (1) DE69702649T2 (en)
WO (1) WO1997029210A2 (en)

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Also Published As

Publication number Publication date
CA2243759A1 (en) 1997-08-14
AU700521B2 (en) 1999-01-07
ATE195004T1 (en) 2000-08-15
WO1997029210A2 (en) 1997-08-14
DE69702649T2 (en) 2001-04-05
AU1856697A (en) 1997-08-28
US5716784A (en) 1998-02-10
DE69702649D1 (en) 2000-08-31
JP3415627B2 (en) 2003-06-09
EP0870063B1 (en) 2000-07-26
CA2243759C (en) 2002-05-21
JPH11506020A (en) 1999-06-02
EP0870063A2 (en) 1998-10-14

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