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Method for detecting point mutations in DNA utilizing fluorescence energy transfer

United States Patent

June 19, 2001
View the Complete Patent at the US Patent & Trademark Office
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.
Parkhurst; Lawrence J. (Lincoln, NE), Parkhurst; Kay M. (Lincoln, NE), Middendorf; Lyle (Lincoln, NE)
Board of Regents of University of Nebraska (Lincoln, NE)
08/ 958,809
October 22, 1997
This invention relates to the identification of genetic mutations and, more particularly, to a method of detecting point mutations in DNA utilizing fluorescence energy transfer. This invention was supported by National Institutes for Health grant DK36288 to the Center for Biotechnology, University of Nebraska-Lincoln.