A homogeneous fluorescence assay for PCR amplicons: its application to real-time, single-tube genotyping.

We have developed a method whereby a single TaqMan probe can be used for many PCR reactions. We demonstrate its application as an integrated system for the direct measurement of allele-specific amplicon generation coupled to the suppression of primer-dimer accumulation in PCR. The system uses a 5'-exonuclease assay of amplicon annealed fluorogenic probes that operates in conjunction with the Amplification Refractory Mutation System, whereby relative changes in reporter fluorescent emission are monitored in real-time using an analytical thermal cycler. 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 DNA sequence or a single pair of probes for genotyping any bi-allelic polymorphism. Three-STAR is, therefore, particularly useful for the single-tube genotype analysis of a variety of human DNA polymorphisms and mutations.