Efficient 12-mutation testing in the CFTR gene: a general model for complex mutation analysis

The identification of the cystic fibrosis transmembrane conductanceregulator (CFTR) gene has led to the identification of morethan 225 presumed disease-causing mutations at the locus. Thediagnosis of cystic fibrosis or the carrier state by directDNA analysis is hindered by this large number. A practical assaymust be able to detect enough mutations to achieve clinicallysignificant sensitivity. The use of allele-specifk oligonucleotideprobes is the most promising of the available methods. However,to date this has generally involved tedious probe-by-probe hybridizations,due to variations in the oligonucleotides' denaturation temperaturescaused by differences in their G-C base-pair content. We havedeveloped a rapid, cost-effective assay that simultaneouslydetects 12 CFTR mutations after multiplex polymerase-chain-reactionamplification of genomic DNA. The test may be readily extendedto detect additional mutations at minimal increase in the costper test or the turnaround time. We improve specificity andavoid the need for individual hybridizations by the use of tetramethylammoniumchloride to virtually eliminate the effects of G-C differences.Coupled with non-invasive sample-collection methods, this isan immediately practical assay for cystic fibrosis. More generally,it will serve as a model for the development of diagnostic testsin other genetic disorders involving complex mutation analysis.