SELECTING DNA FRAGMENTS FOR MUTATION DETECTION BY TEMPERATURE-GRADIENT GEL-ELECTROPHORESIS - APPLICATION TO THE P53 GENE CDNA

Temperature gradient get electrophoresis (TGGE) and related methods ar e widely employed to detect mutations in DNA fragments. DNA melting ma p calculations and GC clamps have been used to enhance the detection o f mutations. While generally successful, these methods have not always revealed base changes within a DNA fragment. Previous work suggested that mutations are detected if they are in a DNA's first melting domai n, and the melting domain is well separated from final strand dissocia tion. Two criteria from the DNA melting theory were established to det ermine when both of these conditions are met. The criteria involve cal culating the derivative melting curve as well as the melting map of a DNA sequence. The approach was applied to the cDNA sequence of the hum an p53 gene. Mutations in the p53 gene are common in human cancers and are generally located in four hot spot regions. Calculations indicate d that three DNA fragments are needed to detect base substitutions in the four hot-spot regions. Predicted melting behavior was experimental ly tested with eight single base substitutions distributed among the f our hot-spot regions. All mutations tested behaved as predicted and we re detected by vertical TGGE. Heteroduplex DNAs formed by melting and reannealing various ratios of wild type and mutant DNA fragments were also examined. Results indicated that point mutations can be detected by ethidium bromide staining from samples containing 10% mutant and 90 % wild-type sequences.