DEVELOPMENT OF RNA-SSCP PROTOCOLS FOR THE IDENTIFICATION AND SCREENING OF CFTR MUTATIONS - IDENTIFICATION OF 2 NEW MUTATIONS

A strategy is described that allows a rapid and accurate identification and screening of cystic fibrosis gene mutations. It consists of setting up and developing RNA single strand conformation polymorphism (rSSCP) protocols, a technique based on the large repertoire of secondary structure of single- stranded RNA. By incorporating the T7 phage promoter sequence into PCR primers, it is possible to carry out rSSCP and compare it to standard single strand conformation polymorphism (SSCP). Several parallel tests indicate that rSSCP detects a higher fraction of single base changes, and is less time consuming than SSCP since it requires only one fairly short electrophoretic run. Using this technique we were able to identify two new Splicing mutations in introns 5 (711+5G-->A) and 10 (1717-8G-->A) of the CFTR gene. (C) 1994 Wiley-Liss, Inc.