Simultaneous detection of two cystic fibrosis alleles using dual-label time-resolved fluorometry

A simple dual-label hybridization test for normal and mutant cystic fibrosis (CF) alleles is described. The assay is based on time-resolved fluorometry (TRF), which allows the simultaneous detection of DNA probes labelled with different lanthanides from one hybridization reaction. DNA was liberated from dried blood disks, normally used in neonatal screening programmes, by boiling in alkaline solution. A 138 bp region including the site of deletion, delta F-508, which is present on about 70% of cystic fibrosis chromosomes, was amplified using the polymerase chain reaction (PCR). The presence or absence of normal and mutant alleles was then determined in a solution hybridization using allele specific oligonucleotide probes labelled either with europium (Eu) or with samarium (Sm) chelates. A common biotinylated probe was used for binding the hybrids onto microtitration wells coated with streptavidin. Some 5 x 10(7) molecules of the normal allele (Eu) and 5 x 10(8) molecules of the mutant allele (Sm) could be detected simultaneously in a single hybridization reaction. The assay was simple to perform and made it possible to reduce the number of hybridizations needed to interpret the sample as being normal, carrier or mutant with regard to the mutation, delta F-508.