SNAPBACK SSCP ANALYSIS - ENGINEERED CONFORMATION CHANGES FOR THE RAPID TYPING OF KNOWN MUTATIONS

Several approaches may be applied to detect known mutations, including restriction enzyme cleavage, allele-specific oligonucleotide (ASO) hy bridization or amplification, dideoxy fingerprinting, and direct DNA s equencing. All these approaches require several extra steps after PCR and may involve radioactive isotopes, time-consuming hybridization, te mplate purification, or digestion steps. The ease and simplicity of th e SSCP test make it a popular choice for mutation detection, but a sig nificant limitation is that some DNA changes will not alter the overal l conformation of either single strand and are thus not amenable to SS CP typing. We describe Snapback SSCP to genotype normal and mdx mice ( an animal model of Duchenne muscular dystrophy) that previously could not be differentiated by conventional SSCP analysis. A snapback primer was designed with additional bases at the 5' terminus, which were com plementary to the normal sequence flanking the mdx mutation and used u nder the original amplification conditions. Each single strand of thes e snapback PCR products now had one terminus capable of re-annealing o r ''snapping back'' to the normal sequence but not the mdx mutation. I n this manner, a conformation change was engineered into the normal st rand that could be readily distinguished from the mdx allele on a SSCP gel. This approach could be applied to the routine screening of other known mutations that are not amenable to detection by simple SSCP ana lysis. (C) 1998 Wiley-Liss, Inc.