RapidF508delandF508CAssay Using Fluorescent Hybridization Probes

Abstract

Amplification and fluorescent genotyping of the cystic fibrosis F508del locus was achieved from human genomic DNA in less than 30 min. The hybridization of adjacent fluorescent probes at the mutation site was monitored by resonance energy transfer between fluorescein and Cy5 during heating or cooling. Characteristic curves were obtained for each genotype; the first derivative of these fluorescent curves has a maximum at an apparent hybridization temperature (T_m) that is specific for each probe/allele duplex. The direction and rate of temperature change determines the difference between the apparent T_m and the true equilibrium T_m. One hundred and five sample were genotyped for the F508del cystic fibrosis mutation by heating and cooling curve profiles. These genotypes were validated by allele-specific amplification. Two fluorescein hybridization probes were designed to match the wild-type sequence perfectly from either codons 502 to 513 or from 504 to 511 on the cystic fibrosis transconductance regulator gene of chromosome 7. While genotyping for the F508del, an allele with the F508C base change was detected. For both F508del and F508C variants, the T_m shift from wild type was greater with a 24-mer probe than with a 35-mer probe. Fluorescent monitoring of hybridization probes is a versatile technique that can detect unexpected sequence alterations.