Simple triple-label detection of seven cystic fibrosis mutations by time-resolved fluorometry.
We describe a simple hybridization assay performed in microtitration wells with use of DNA probes labeled with three different lanthanide chelates for detection of seven mutations that cause cystic fibrosis. The assay is based on DNA amplification of four fragments containing the mutations (delta F508, G1717-->A, G542X, R553X, 3905 insertion T, W1282X, and N1303K) by PCR, followed by hybridization with short, allele-specific oligonucleotide probes labeled with europium, terbium, or samarium chelates. Because the technology makes it possible to hybridize three DNA probes simultaneously in one reaction, all 14 mutation-related alleles were detected in a total of five reaction wells. Blood spot specimens, obtained from children with cystic fibrosis, their parents, and their siblings, have been assayed, and for all the probes the positive signal-to-noise ratios are > 10. Solution hybridization utilizing triple-label time-resolved fluorometry combined with PCR is a suitable procedure for large-scale screening and automation.
Heinonen
P
P
Department of Biotechnology, University of Turku, Finland. pia.heinonen@utu.fi
Iitiä
A
A
Torresani
T
T
Lövgren
T
T
D000483:Alleles; D002614:Chelating Agents; D003550:Cystic Fibrosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D004247:DNA / Q000032:analysis*; D015342:DNA Probes; D005063:Europium; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D008674:Metals, Rare Earth; D009154:Mutation; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D012493:Samarium; D013725:Terbium
C505032:CFTR protein, human; D002614:Chelating Agents; D015342:DNA Probes; D008674:Metals, Rare Earth; D013725:Terbium; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D012493:Samarium; D005063:Europium; D004247:DNA