Title: Method for detecting point mutations in DNA utilizing fluorescence energy transfer

Abstract

A method for detecting point mutations in DNA using a fluorescently labeled oligomeric probe and Forster resonance energy transfer (FRET) is disclosed. The selected probe is initially labeled at each end with a fluorescence dye, which act together as a donor/acceptor pair for FRET. The fluorescence emission from the dyes changes dramatically from the duplex stage, wherein the probe is hybridized to the complementary strand of DNA, to the single strand stage, when the probe is melted to become detached from the DNA. The change in fluorescence is caused by the dyes coming into closer proximity after melting occurs and the probe becomes detached from the DNA strand. The change in fluorescence emission as a function of temperature is used to calculate the melting temperature of the complex or T.sub.m. In the case where there is a base mismatch between the probe and the DNA strand, indicating a point mutation, the T.sub.m has been found to be significantly lower than the T.sub.m for a perfectly match probelstand duplex. The present invention allows for the detection of the existence and magnitude of T.sub.m, which allows for the quick and accurate detection of a point mutation in the DNA strand and, inmore » some applications, the determination of the approximate location of the mutation within the sequence. « less

Inventors:

Parkhurst, Lawrence J ^[1]; Parkhurst, Kay M ^[1]; Middendorf, Lyle ^[1]

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1. Lincoln, NE

Issue Date:

Mon Jan 01 00:00:00 EST 2001

Research Org.:

Center for Biotechnology, University of Nebraska-Lincoln

OSTI Identifier:

873795

Patent Number(s):

6248518

Assignee:

Board of Regents of University of Nebraska (Lincoln, NE)

Patent Classifications (CPCs):

C - CHEMISTRY C12 - BIOCHEMISTRY C12Q - MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS

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C - CHEMISTRY C12 - BIOCHEMISTRY C12Q - MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS

C12Q1/6818 - involving interaction of two or more labels, e.g. resonant energy transfer
C12Q1/6827 - for detection of mutation or polymorphism
C12Q2527/107 - Temperature of melting, i.e. Tm
C12Q2565/1015 - labels being on the same oligonucleotide
C12Q2565/107 - Alteration in the property of hybridised versus free label oligonucleotides

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Resource Type:

Patent

Country of Publication:

United States

Language:

English

Subject:

method; detecting; mutations; dna; utilizing; fluorescence; energy; transfer; fluorescently; labeled; oligomeric; probe; forster; resonance; fret; disclosed; selected; initially; dye; donor; acceptor; pair; emission; dyes; changes; dramatically; duplex; stage; hybridized; complementary; strand; single; melted; detached; change; caused; coming; closer; proximity; melting; occurs; function; temperature; calculate; complex; base; mismatch; indicating; mutation; found; significantly; perfectly; match; probelstand; allows; detection; existence; magnitude; quick; accurate; applications; determination; approximate; location; sequence; resonance energy; dna strand; melting temperature; energy transfer; fluorescence emission; accurate detection; selected probe; complementary strand; fluorescently labeled; acceptor pair; single strand; approximate location; dna utilizing; /435/999/