title: Fluorescence-based oligonucleotide ligation assay for analysis of cystic fibrosis transmembrane conductance regulator gene mutations.
abstract: 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.
authors.affiliation: Applied Biosystems Division, Perkin-Elmer Corporation, Foster City, California 94404, USA.
authors.forename: F A
authors.initials: FA
authors.lastname: Eggerding
authors.forename: D M
authors.initials: DM
authors.lastname: Iovannisci
authors.forename: E
authors.initials: E
authors.lastname: Brinson
authors.forename: P
authors.initials: P
authors.lastname: Grossman
authors.forename: E S
authors.initials: ES
authors.lastname: Winn-Deen
mesh_terms: D000483:Alleles; D001483:Base Sequence; D003550:Cystic Fibrosis / Q000175:diagnosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA / Q000235:genetics*; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000737:chemistry; D005809:Genes, Regulator / Q000235:genetics*; D006801:Humans; D008025:Ligases / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000737:chemistry* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: C505032:CFTR protein, human; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D015345:Oligonucleotide Probes; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA; D008025:Ligases