Allelic discrimination by nick-translation PCR with fluorgenic probes

TLDR

Nick‑translation PCR was performed using two fluorogenic probes—one targeting the normal F508 codon and the other the ΔAF508 deletion—each labeled with a distinct fluorescent dye and a quencher, and the probes were incorporated at the start of the PCR and degraded during thermal cycling. The probes produced fluorescence only when perfectly complementary, allowing the target DNA to be identified from the post‑PCR fluorescence emission spectrum.

Abstract

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 complementary to a sequence containing the AF508 three base pair deletion ( mut DNA). Each probe contained a unique and spectrally resolvable fluorescent Indicator dye at the 5′ end and a common quencher dye attached to the seventh nucleotide from the 5′ end. The F508/ΔAF508 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 and were degraded during thermal cycling. Although both probes were degraded, each probe generated fluorescence from its indicator dye only when the sequence between the indicator and quencher dyes was perfectly complementary to target. The identity of the target DNA could be determined from the post-PCR fluorescence emission spectrum.