WO2001066799A3 - Real time quantitative pcr with intercalating dye for single and multiplex target dna - Google Patents

Real time quantitative pcr with intercalating dye for single and multiplex target dna Download PDF

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Publication number
WO2001066799A3
WO2001066799A3 PCT/US2001/007101 US0107101W WO0166799A3 WO 2001066799 A3 WO2001066799 A3 WO 2001066799A3 US 0107101 W US0107101 W US 0107101W WO 0166799 A3 WO0166799 A3 WO 0166799A3
Authority
WO
WIPO (PCT)
Prior art keywords
target
melting
fms
tms
slope
Prior art date
Application number
PCT/US2001/007101
Other languages
French (fr)
Other versions
WO2001066799A2 (en
Inventor
Michael Edward Schaffer
Susan Yen-Tee Tseng
Original Assignee
Du Pont
Michael Edward Schaffer
Susan Yen-Tee Tseng
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 Du Pont, Michael Edward Schaffer, Susan Yen-Tee Tseng filed Critical Du Pont
Priority to AU2001250803A priority Critical patent/AU2001250803A1/en
Priority to US10/204,889 priority patent/US20040053230A1/en
Priority to JP2001565402A priority patent/JP2004533801A/en
Priority to EP01924120A priority patent/EP1358346A2/en
Publication of WO2001066799A2 publication Critical patent/WO2001066799A2/en
Publication of WO2001066799A3 publication Critical patent/WO2001066799A3/en

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Classifications

    • 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

Abstract

The PCR-based, dsDNA quantification method monitors the fluorescence of a target, whose melting characteristics is predetermined, during each amplification cycle at selected time-points. Fluorescence is measured immediately after the annealing phase (FE at TE), immediately below (FMS at TMS) and above (FME at TME) the melting of the target/amplicon. A change in slope from a baseline slope (SB = -(FMS - FE)/(TMS -TE)) to a melting phase slope (SM = -(FME - FMS)/(TME - TMS) indicates a specific amplification. The number of amplification cycles (CT) it takes for the quantity (SM - SB) to become greater than zero correlates with the starting concentration of the target (C). The concentration of the target in a sample is determined by comparing the value of CT for the sample with a standard curve. By selecting targets with distinguishable melting curve characteristics, multiple targets can be simultaneously detected.
PCT/US2001/007101 2000-03-07 2001-03-07 Real time quantitative pcr with intercalating dye for single and multiplex target dna WO2001066799A2 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU2001250803A AU2001250803A1 (en) 2000-03-07 2001-03-07 Real time quantitative pcr with intercalating dye for single and multiplex target dna
US10/204,889 US20040053230A1 (en) 2001-03-07 2001-03-07 Real time quantitative pcr with intercalating dye for single and multiplex target dna
JP2001565402A JP2004533801A (en) 2000-03-07 2001-03-07 Real-time quantitative PCR using intercalation dyes for single and multiple target DNA
EP01924120A EP1358346A2 (en) 2000-03-07 2001-03-07 Real time quantitative pcr with intercalating dye for single and multiplex target dna

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US18753700P 2000-03-07 2000-03-07
US60/187,537 2000-03-07

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US10/204,889 A-371-Of-International US20040053230A1 (en) 2000-03-07 2001-03-07 Real time quantitative pcr with intercalating dye for single and multiplex target dna
US10/829,430 Continuation US20040175752A1 (en) 2000-03-07 2004-04-21 Real time quantitative PCR with intercalating dye for single and multiplex target DNA

Publications (2)

Publication Number Publication Date
WO2001066799A2 WO2001066799A2 (en) 2001-09-13
WO2001066799A3 true WO2001066799A3 (en) 2003-09-04

Family

ID=22689385

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2001/007101 WO2001066799A2 (en) 2000-03-07 2001-03-07 Real time quantitative pcr with intercalating dye for single and multiplex target dna

Country Status (4)

Country Link
EP (1) EP1358346A2 (en)
JP (1) JP2004533801A (en)
AU (1) AU2001250803A1 (en)
WO (1) WO2001066799A2 (en)

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NZ575481A (en) 2000-10-18 2010-10-29 Pharmasset Inc Simultaneous quantification of nucleic acids in diseased cells
KR100624667B1 (en) * 2000-10-26 2006-09-18 내셔널 푸드 리서치 인스티튜트 Methods of quantitative detection of genetic recombinants and standard molecules for the methods
AU2002218413A1 (en) 2000-11-30 2002-06-11 Ses Europe N.V. Glyphosate resistant transgenic sugar beet characterised by a specific transgene insertion (t227-1), methods and primers for the detection of said insertion
US9169516B2 (en) 2003-01-24 2015-10-27 University Of Utah Research Foundation Methods of predicting mortality risk by determining telomere length
CN1973052B (en) * 2004-06-25 2013-07-24 杜邦公司 DNA sequences for the detection of and differentation amongst pathogenic e.coli
JP4777631B2 (en) * 2004-09-27 2011-09-21 株式会社日立ハイテクノロジーズ Nucleic acid amplification analysis method and apparatus
EP2333561A3 (en) 2005-03-10 2014-06-11 Gen-Probe Incorporated System for performing multi-formatted assays
EP2183394B1 (en) * 2007-09-07 2012-12-12 Third Wave Technologies, Inc. Methods and applications for target quantification
EP2215265B1 (en) * 2007-11-14 2013-05-22 Grifols Therapeutics Inc. Method for adjusting results of a polymerase chain reaction (pcr) instrument
EP2163239A1 (en) * 2008-05-27 2010-03-17 Qiagen GmbH Products containing bioparticles, method for their manufacture
JP5840950B2 (en) 2008-12-22 2016-01-06 ユニバーシティ・オブ・ユタ・リサーチ・ファウンデイション Monochromatic multiplex quantitative PCR
US8606527B2 (en) 2009-02-27 2013-12-10 Bio-Rad Laboratories, Inc. SNP detection by melt curve clustering
EP2524053A1 (en) * 2010-01-15 2012-11-21 Steffen Mergemeier Method for detecting more than one target in a pcr-based approach applying an unspecific dye which is not interfering with the emission of fluorophore-labeled probes
US9932628B2 (en) 2012-07-27 2018-04-03 Gen-Probe Incorporated Dual reference calibration method and system for quantifying polynucleotides
JP6546158B2 (en) 2013-05-22 2019-07-17 テロメア ダイアグノスティクス インコーポレイテッド Measurement of short telomere abundance
EP3230475A4 (en) 2014-12-09 2018-07-18 Seegene, Inc. Differentiation of signals for target nucleic acid sequences
AU2015372584A1 (en) 2014-12-30 2017-06-29 Telomere Diagnostics, Inc. Multiplex quantitative PCR

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1295941B1 (en) * 2000-06-27 2009-12-16 National Institute of Advanced Industrial Science and Technology Novel nucleic acid probes and method of assaying nucleic acid by using the same

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
MORRISON T B ET AL: "Rapid and sensitive quantification of Borrelia burgdorferi-infected mouse tissues by continuous fluorescent monitoring of PCR.", JOURNAL OF CLINICAL MICROBIOLOGY. UNITED STATES APR 1999, vol. 37, no. 4, April 1999 (1999-04-01), pages 987 - 992, XP002210169, ISSN: 0095-1137 *
RIRIE K M ET AL: "PRODUCT DIFFERENTIATION BY ANALYSIS OF DNA MELTING CURVES DURING THE POLYMERASE CHAIN REACTION", ANALYTICAL BIOCHEMISTRY, ACADEMIC PRESS, SAN DIEGO, CA, US, vol. 245, no. 2, 1997, pages 154 - 160, XP001015991, ISSN: 0003-2697 *
See also references of EP1358346A2 *
SHEPHERD M ET AL: "MONITORING OF FLUORESCENCE DURING DNA MELTING AS A METHOD FOR DISCRIMINATION AND DETECTION OF PCR PRODUCTS IN VARIETY IDENTIFICATION", MOLECULAR BREEDING: NEW STRATEGIES IN PLANT IMPROVEMENT, KLUWER ACADEMIC PUBLISHERS, NL, vol. 4, no. 6, 1998, pages 509 - 517, XP001016033, ISSN: 1380-3743 *

Also Published As

Publication number Publication date
EP1358346A2 (en) 2003-11-05
WO2001066799A2 (en) 2001-09-13
AU2001250803A1 (en) 2001-09-17
JP2004533801A (en) 2004-11-11

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