WO2001094638A8 - Asynchronous primed pcr - Google Patents

Asynchronous primed pcr

Info

Publication number
WO2001094638A8
WO2001094638A8 PCT/US2001/018464 US0118464W WO0194638A8 WO 2001094638 A8 WO2001094638 A8 WO 2001094638A8 US 0118464 W US0118464 W US 0118464W WO 0194638 A8 WO0194638 A8 WO 0194638A8
Authority
WO
WIPO (PCT)
Prior art keywords
probe
asynchronous
reporter dye
stranded
annealed
Prior art date
Application number
PCT/US2001/018464
Other languages
French (fr)
Other versions
WO2001094638A2 (en
WO2001094638A3 (en
Inventor
Caifu Chen
Michael Egholm
Lawrence Haff
Original Assignee
Applera 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 Applera Corp filed Critical Applera Corp
Priority to DE60140487T priority Critical patent/DE60140487D1/en
Priority to EP01946152A priority patent/EP1368489B1/en
Priority to AT01946152T priority patent/ATE448325T1/en
Priority to JP2002502178A priority patent/JP2004509613A/en
Priority to CA002412413A priority patent/CA2412413A1/en
Priority to AU6823501A priority patent/AU6823501A/en
Publication of WO2001094638A2 publication Critical patent/WO2001094638A2/en
Publication of WO2001094638A8 publication Critical patent/WO2001094638A8/en
Publication of WO2001094638A3 publication Critical patent/WO2001094638A3/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions
    • C12Q1/686Polymerase chain reaction [PCR]

Abstract

An asynchronous thermal cycling protocol for nucleic acid amplification uses two primers with thermal melting temperatures different by about 10 to 30 °C. After the higher melting primer has annealed and polymerase mediated extension, the uncopied, single-stranded target sequence may be hybridized and detected by a probe. DNA probes may be cleaved by the exonuclease activity of a polymerase. The probe may be a non-cleaving analog such as PNA. When a probe is labelled with a reporter dye and a quencher selected to undergo energy transfer, e.g. FRET, fluorescence from the reporter dye may be effectively quenched when the probe is unbound. Upon hybridization of the probe to complementary target or upon cleavage while bound to target, the reporter dye is no longer quenched, resulting in a detectable amount of fluorescence. The second, lower-melting primer may be annealed and extended to generate a double-stranded nucleic acid. Amplification may be monitored in real time, including each cycle, or at the end point. The asynchronous PCR thermal cycling protocol can generate a preponderance of the PCR amplicon in single-stranded form by repetition at the end of the protocol of annealing and extension of the higher melting primer.
PCT/US2001/018464 2000-06-06 2001-06-06 Asynchronous primed pcr WO2001094638A2 (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
DE60140487T DE60140487D1 (en) 2000-06-06 2001-06-06 Asynchrones pcr priming
EP01946152A EP1368489B1 (en) 2000-06-06 2001-06-06 Asynchronous primed pcr
AT01946152T ATE448325T1 (en) 2000-06-06 2001-06-06 ASYNCHRONOUS PCR PRIMING
JP2002502178A JP2004509613A (en) 2000-06-06 2001-06-06 Asynchronous stimulus PCR
CA002412413A CA2412413A1 (en) 2000-06-06 2001-06-06 Asynchronous primed pcr
AU6823501A AU6823501A (en) 2000-06-06 2001-06-06 Asynchronous primed pcr

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US20988300P 2000-06-06 2000-06-06
US60/209,883 2000-06-06
US09/875,211 US6887664B2 (en) 2000-06-06 2001-06-05 Asynchronous primed PCR
US09/875,211 2001-06-05

Publications (3)

Publication Number Publication Date
WO2001094638A2 WO2001094638A2 (en) 2001-12-13
WO2001094638A8 true WO2001094638A8 (en) 2002-04-11
WO2001094638A3 WO2001094638A3 (en) 2003-07-10

Family

ID=26904613

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2001/018464 WO2001094638A2 (en) 2000-06-06 2001-06-06 Asynchronous primed pcr

Country Status (8)

Country Link
US (2) US6887664B2 (en)
EP (3) EP2316975A1 (en)
JP (1) JP2004509613A (en)
AT (1) ATE448325T1 (en)
AU (1) AU6823501A (en)
CA (1) CA2412413A1 (en)
DE (1) DE60140487D1 (en)
WO (1) WO2001094638A2 (en)

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Also Published As

Publication number Publication date
US20030207266A1 (en) 2003-11-06
WO2001094638A2 (en) 2001-12-13
EP1368489B1 (en) 2009-11-11
WO2001094638A3 (en) 2003-07-10
AU6823501A (en) 2001-12-17
CA2412413A1 (en) 2001-12-13
ATE448325T1 (en) 2009-11-15
US6887664B2 (en) 2005-05-03
DE60140487D1 (en) 2009-12-24
EP2256213A1 (en) 2010-12-01
EP1368489A2 (en) 2003-12-10
EP2316975A1 (en) 2011-05-04
JP2004509613A (en) 2004-04-02
US20050130178A1 (en) 2005-06-16

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