Fluorescence-based oligonucleotide ligation assay for analysis of cystic fibrosis transmembrane conductance regulator gene mutations

• Published in: Human mutation Vol. 5; no. 2; p. 153
• Main Authors: Eggerding, F A, Iovannisci, D M, Brinson, E, Grossman, P, Winn-Deen, E S
• Format: Journal Article
• Language: English
• Published: United States 1995
• Subjects: Alleles; Base Sequence; Cystic Fibrosis - diagnosis; Cystic Fibrosis - genetics; Cystic Fibrosis Transmembrane Conductance Regulator; DNA - genetics; DNA Mutational Analysis; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes - chemistry; Genes, Regulator - genetics; Humans; Ligases - chemistry; Ligases - metabolism; Membrane Proteins - genetics; Molecular Sequence Data; Oligonucleotide Probes - chemistry; Oligonucleotide Probes - metabolism; Polymerase Chain Reaction; Sensitivity and Specificity
• ISSN: 1059-7794
• DOI: 10.1002/humu.1380050209

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 delta F508 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.