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    I11111111111111I11I11S000IIIIIIIII740701~1111111111IIIII111111111111111111(12) United States Patent ( i o ) Patent No.: US 6,174,670 B1Wittwer et al. (45) Date ofPatent: Jan. 16,2001

    MONITORING AMPLIFICATION OF DNADURING PCR

    Inventors: Carl T. Wittwer, Salt Lake City, UT

    (US); Kirk M. Ririe, Idaho Falls, ID(US); Randy P. Rasmussen, Salt LakeCity, UT (US)

    Assignee: University of Utah ResearchFoundation, Salt Lake City, UT (US)

    Under 35 U.S.C. 154(b), the term of thispatent shall be extended for 0 days.

    Notice:

    Appl. No.: 081869,276

    Filed: Jun. 4, 1997

    Related U.S. Application Data

    Continuation-in-part ofapplication No. 081818,267, filed onMar. 17,1997, which is a continuation-in-part ofapplicationNo. 081658,993, filed on Jun. 4, 1996, now abandoned.Int. Cl? ............................... C12Q 1/68; C12P 19134U.S. C1. ................................. 43516; 435191.2; 436194Field of Search .......................... 43516, 91.2; 436194

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    Primary E x a m i n e r x e n n e t h R. Horlick(74) Attorney, Agent, or F i r m a a r n e s & Thornburg(57) ABSTRACTMethods of monitoring hybridization during polymerasechain reaction are disclosed. These methods are achievedwith rapid thermal cycling and use of double stranded DNAdyes or specific hybridization probes. A fluorescence reso-nance energy transfer pair comprises fluorescein and Cy5 orCy5.5. Methods for quantitating amplified DNA and deter-mining its purity are carried out by analysis of melting and

    reannealing curves.

    107 Claims, 52 Drawing Sheets

    Microfiche Appendix Included(1 Microfiche, 54 Pages)

    10

    Fluorescence RatioAbove Background(1og) 1 +Labeled Primer with singleHybridizationProbe

    0.10 10 20 30 40

    Cycle

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    US 6,174,670 B1Pane 2

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    Higuchi, R., et al., Kinetic PCR Analysis: Real-time Moni-toring of DNA Amplification Reactions, BioiTechnology,vol. 11, pp. 1026-1030 (1993).Hillen, W., et al., High Resolution Experimental and Theo-retical Thermal Denaturation Studies on Small OverlappingRestriction Fragments Containing theEscherichia coli Lac-tose Genetic Control Region, The Journal ofBiologicalChemistry, vol. 256, No. 6, pp. 2761-2766 (1981).Hiyoshi, M., et al., Assay of DNA Denaturation by Poly-merase Chain Reaction-Driven Fluorescence ResonanceEnergy Transfer, Analytical Biochemistry,vol. 221, pp.306-311 (1994).Hoffman, L.M., et al., use of a Gas Chromatograph Ovenfor DNAAmplification by the Polymerase Chain Reaction,

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    0 566 7510 580 362 A10 640 828A1

    0 636 4130 643 140

    0 674 0090 686 6992 122 18760212 986

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    *

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