A HOMOGENEOUS FLUORESCENCE ASSAY FOR PCR AMPLICONS - ITS APPLICATION TO REAL-TIME, SINGLE-TUBE GENOTYPING

We have developed a method whereby a single Taq-Man(TM) probe can be u sed for many PCR reactions. We demonstrate its application as an integ rated system for the direct measurement of allele-specific amplicon ge neration coupled to the suppression of primer-dimer accumulation in PC R. The system uses a 5'-exonuclease assay of amplicon annealed fluorog enic probes that operates in conjunction with the Amplification Refrac tory Mutation System, whereby relative changes in reporter fluorescent emission are monitored in real-time using an analytical thermal cycle r. We have called this system Three-STAR, and it is universal in that it can either use a single probe for the detection of any one target D NA sequence or a single pair of probes for genotyping any bi-allelic p olymorphism. Three-STAR is, therefore, particularly useful for the sin gle-tube genotype analysis of a variety of human DNA polymorphisms and mutations.