Method for detecting point mutations in DNA utilizing fluorescence energy transfer

Parkhurst, Lawrence J; Parkhurst, Kay M; Middendorf, Lyle

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

Patent · Mon Jan 01 00:00:00 EST 2001

OSTI ID:873795

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

Lincoln, NE

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, in some applications, the determination of the approximate location of the mutation within the sequence.

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Research Organization:: Center for Biotechnology, University of Nebraska-Lincoln

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

Patent Number(s):: US 6248518

OSTI ID:: 873795

Country of Publication:: United States

Language:: English

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

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