Simple triple-label detection of seven cystic fibrosis mutations by time-resolved fluorometry
Heinonen, P.; Iitiä, A.; Torresani, T.; Lövgren, T.
Clinical Chemistry 43(7): 1142-1150
1997
ISSN/ISBN: 0009-9147 PMID: 9216449 Document Number: 481112
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, G-1717 fwdarw 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 gt 10. Solution hybridization utilizing triple-label time-resolved fluorometry combined with PCR is a suitable procedure for large-scale screening and automation.