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Fluorescence‐based oligonucleotide ligation assay for analysis of cystic fibrosis transmembrane conductance regulator gene mutations
Author(s) -
Eggerding Faye A.,
Iovannisci David M.,
Brinson Eleanor,
Grossman Paul,
WinnDeen Emily S.
Publication year - 1995
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.1380050209
Subject(s) - biology , microbiology and biotechnology , oligonucleotide , cystic fibrosis transmembrane conductance regulator , multiplex ligation dependent probe amplification , mutant , ligation , mutation , multiplex , exon , multiplex polymerase chain reaction , gene , genetics , point mutation , oligomer restriction , polymerase chain reaction
Isolation of the gene for cystic fibrosis (CF), the cystic fibrosis transmembrane conductance regulator (CFTR), provided a basis for analyzing its molecular pathology and resulted in the identification of < 400 mutations associated with disease. Except for the ΔF508 mutation, no other single mutation accounts for > 5% of CF chromosomes in most populations, and most mutation frequencies are < 1%. A strategy based on multiplex PCR followed by multiplex allele‐specific oligonucleotide probe ligation was used to detect 30 mutations, distributed throughout ten exons and seven introns of the CFTR gene, that together account for > 96% of CF mutant chromosomes worldwide. Mutations were detected by competitive oligonucleotide probe ligation to detect normal and/or mutant genotypes in one reaction. Three probes (one common and two allelic probes) were needed for analysis of each mutation. Probes hybridized to target DNA were joined by a thermostable ligase if there were no mismatches at their junctions; temperature cycling resulted in a linear increase in product. Common probes were labeled with fluorochromes, and allelic probes each had different lengths. Ligation products were analyzed electrophoretically on a fluorescent DNA sequencer. The results show that combined PCR and probe ligation amplification rapidly and reliably screen for CF homozygotes and carriers. © Wiley‐Liss, Inc.