GENOTYPING WITH MOLECULAR BEACONS: USING FLUORESCENCE TO RAPIDLY DETECT TARGET SEQUENCES IN SOLUTION

Bodega Marine Laboratory, University of California, 2099 Westside Rd., Bodega Bay, Ca 94923 USA

We are developing molecular and statistical methods to identify endangered salmon stocks for management and conservation. California's Central Valley watershed has four runs of chinook salmon (winter, spring, fall and late fall). The winter-run population suffered precipitous declines in recent years and in 1994 was federally listed as endangered. Effective management requires the ability to discriminate among the runs, but overlapping run timing, coupled with morphological similarities, confound field identification. To this end, we developed a suite of fluorescently labeled microsatellite markers that enable run discrimination based on allele frequencies, but more markers are needed. Recently, we adopted a novel technique using molecular beacons (MB) to detect run-diagnostic single nucleotide polymorphisms in the Mhc class II B 1 exon. Molecular beacons are short oligonucleotide probes with a stem-and-loop configuration that fluoresce only when hybridized to a target sequence amplicon. A fluorophore attached to the 5' end and a non-fluorescent quenching moiety bound to the 3' end are held in close proximity through a short segment of complimentary 5' and 3' ends. When hybridized to the target sequence the quencher and fluorophore become disassociated and fluorescence can be detected post-PCR. Genotyping is achieved with three separate PCR reactions, each containing a pair of MBs. Probes identify alternate nucleotides at a given polymorphic site and maximize the instability of probe-target mismatches. Multiplexing is performed by labeling one MB with 6-FAM and the other with TAMRA. The large scanning area and multi-color capabilities of the Hitachi FMBIO II Fluorescence Imaging System allows up to six microtiter plates (up to 2304 samples) to be read simultaneously. This system is amenable to auto-scoring and provides a rapid and inexpensive alternative to traditional membrane based genotyping techniques.