University of Nebraska

Lawrence J Parkhurst

Lyle R Middendorf

Kay M Parkhurst

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 . In the case where there is a base mismatch between the probe and the DNA strand, indicating a point mutation, the T has been found to be significantly lower than the T for a perfectly match probe/stand duplex. The present invention allows for the detection of the existence and magnitude of T , 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.

Probe for detecting point mutations in DNA utilizing fluorescence energy transfer

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,. In the case where there is a base mismatch between the probe and the DNA strand, indicating a point mutation, the T,has been found to be significantly lower than the T,for a perfectly match probelstand duplex. The present invention allows for the detection of the existence and magnitude of T,, 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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Lincoln, NE, United States of America

Lawrence J Parkhurst