Genetic mutations can give rise to variations in alleles, cause genetic diseases, and predispose affected individuals to illnesses. Routine detection of genetic mutations is a time-consuming and tedious task, since most methods require the use of high resolution gel electrophoresis. In contrast, luminescence-based assays are more rapid and easily automated for high throughput operation. In the assays to be developed, detecting mutations of a single nucleotide in regions spanning hundreds of base pairs may be facilitated by resonance energy transfer between appropriate luminescent probes. Problems associated with previous fluorescence assays are circumvented by the use of recently developed probes. While short-lived fluorescence from conventional dyes must compete with high backgrounds from light scattering and autofluorescence of biological specimens, time-resolved fluorescence of lanthanide-based probes eliminate these background problems. Background is reduced further by using hairpin probes that are quenched until hybridized to the correct target sequence. Therefore, the proposed assays may be used directly on samples from patients or PCR-amplified products. The ability to screen large numbers of samples in these microassays will benefit areas of biochip development, tissue typing, gene expression, multiplex sequencing, and genetic disease testing. These enhanced assays will have impacts on health care cost efficiency, early diagnosis, treatment, and prevention of diseases. PROPOSED COMMERCIAL APPLICATIONS: This technology will provide probes which emit sensitive, low-background time-resolved fluorescence signals upon hybridization to targets, making it ideal for biochip applications for simultaneous microsampling of sequence and detection of mutations. Applications include identification of genetic diseases, tissue typing of alleles, and multiplex hybridization sequencing.
