Methods for analysis of multiple cystic fibrosis mutations |
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Authors: | Ivy S L Ng Robert Pace Michael V Richard Keiko Kobayashi Bat-sheva Kerem Lap-Chee Tsui Arthur L Beaudet |
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Institution: | (1) Baylor College of Medicine, Institute for Molecular Genetics, USA;(2) Howard Hughes Medical Institute, 77030 Houston, TX, USA;(3) Department of Genetics, The Hospital for Sick Children, M5G 1X8 Toronto, Ontario, Canada;(4) Baylor College of Medicine, Howard Hughes Medical Institute, 77030 Houston, TX, USA;(5) Present address: Department of Genetics, Hebrew University of Jerusalem, 91904 Jerusalem, Israel |
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Abstract: | Summary A large number of mutations causing cystic fibrosis (CF) have been reported. In an attempt to improve methods for genetic diagnosis and for heterozygote screening, we evaluated methods for efficient analysis of the F508, G542X, G551D, R553X, and N1303K mutations. We found that multiple mutations can be analyzed simultaneously using hybridization with allelespecific oligonucleotides. Alternatively all of these mutations can be detected by amplification of DNA followed by restriction enzyme digestion and analysis on polyacrylamide gels. A previously reported method for use of modified primers for DNA amplification to allow detection of virtually any single-base change by restriction enzyme analysis proved particularly useful. The common F508 mutation and three mutations in exon 11 were analyzed using a multiplex amplification reaction followed by double digestion with restriction enzymes and electrophoresis in a single lane on a polyacrylamide gel. In a sample of 439 CF chromosomes from North American Caucasians, the frequencies of various mutations were as follows: F508=75.8%, G542X=2.7%, G551D=3.2%, R553X=1.4%, and N1303K=1.4% for a total of 84.5% detection of CF chromosomes by analysis for these five mutations. |
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