ALLELIC DISCRIMINATION BY NICK-TRANSLATION PCR WITH FLUOROGENIC PROBES

Nick-translation PCR was performed with fluorogenic probes. Two probes were used: one complementary to a sequence containing the F508 codon of the normal human cystic fibrosis (CF) gene (wt DNA) and one complem entary to a sequence containing the DELTAF508 three base pair deletion (mut DNA). Each probe contained a unique and spectrally resolvable fl uorescent indicator dye at the 5' end and a common quencher dye attach ed to the seventh nucleotide from the 5' end. The F508/DELTAF508 site was located between the indicator and quencher. The probes were added at the start of a PCR containing mut DNA, wt DNA or heterozygous DNA a nd were degraded during thermal cycling. Although both probes were deg raded, each probe generated fluorescence from its indicator dye only w hen the sequence between the indicator and quencher dyes was perfectly complementary to target. The identity of the target DNA could be dete rmined from the post-PCR fluorescence emission spectrum.