Denaturing gradient gel analysis of single-base substitutions at a mouse adenine phosphoribosyltransferase splice...

Denaturing gradient gel electrophoresis (DGGE) can detect single-base changes in DNA. We used site-directed mutagenesis to produce all six possible single-base substitutions at a splice acceptor consensus AG dinucleotide within the mouse adenine phosphoribosyltransferase (aprt) gene. Studies of mouse and Chinese hamster cell aprt indicate a high level of both spontaneous and induced mutations in this region. We systematically evaluated each of the six mutations by DGGE. Five of the six mutant sequences could be distinguished from wildtype by DGGE analysis of a 560-bp fragment containing the mutation. However, one mutant could not be distinguished from wild-type, and some of the mutant constructs could not be distinguished from each other. Analysis of DNA heteroduplexes consisting of wild-type and mutant strands or two different mutant strands enabled all mutant constructs to be distinguished from wild-type and from each other. The pairwise mixtures resulted in 24 different heteroduplexes, all of which were less stable than the parental homoduplexes. End labeling of DNA prior to heteroduplex formation and subsequent DGGE analysis enabled us to determine the relative destabilization caused by different types of single-base mismatches.