Author/Editor		Ravnik-Glavač, M; Glavač, D; Dean, M
Title		Sensitivity of single-strand conformation polymorphism and heteroduplex method for mutation detection in the cystic fibrosis gene
Type		članek
Source		Hum Mol Genet
Vol. and No.		Letnik 3, št. 5
Publication year		1994
Volume		str. 801-7
Language		eng
Abstract		The gene responsible for cystic fibrosis (CF) contains 27 coding exons and more than 300 independent mutations have been identified. An efficient and optimized strategy is required to identify additional mutations and/or to screen patient samples for the presence of known mutations. We have tested several different conditions for performing single-stranded conformation polymorphism (SSCP) analysis in order to determine the efficiency of the method and to identify the optimum conditions for mutation detection. Each exon and corresponding exon boundaries were amplified. A panel of 134 known CF mutations were used to test the efficiency of detection of mutations. The SSCP conditions were varied by altering the percentage and cross-linking of the acrylamide, employing MDE (an acrylamide substitute), and by adding sucrose and glycerol. The presence of heteroduplexes could be detected on most gels and in some cases contributed to the ability to distinguish certain mutations. Each analysis condition detected 75-98 percent of the mutations, and all of the mutations could be detected by at least one condition. Therefore, an optimized SSCP analysis can be used to efficiently screen for mutations in a large gene.
Descriptors		CYSTIC FIBROSIS DNA MUTATIONAL ANALYSIS MEMBRANE PROTEINS NUCLEIC ACID HYBRIDIZATION POLYMORPHISM (GENETICS) BASE SEQUENCE DNA, SINGLE-STRANDED EXONS GENES, STRUCTURAL GENETIC SCREENING MOLECULAR SEQUENCE DATA MUTATION NUCLEIC ACID CONFORMATION SENSITIVITY AND SPECIFICITY