Efficient Mutation Detection in Mismatch Repair Genes Using a Combination of Single-Strand Conformational Polymorphism and Heteroduplex Analysis at a Controlled Temperature

Single-strand conformational polymorphism analysis (SSCP) and heteroduplex analysis (HD) were tested as methods for mutation screening with respect to experimental variation, sensitivity, and specificity. Thirty-nine fluorescently labeled PCR products covering the two mismatch repair genes, hMLH1 and hMSH2, were tested in 15 patients for pattern changes, using SSCP and HD at two temperatures, in a total of 2340 runs. SSCP was most efficient in detecting base changes, whereas HD was the method of choice when detecting deletions. SSCP and HD at 20°C were most effective (sensitivity 97%, specificity 49%), and SSCP and HD at 10°C gave no additional information, except in one case where an exon had two base changes. Several mutations only showed a small pattern change in one of the two strands, most explicit at 20°C. No correlation between the type of base change and the size or direction of the pattern changes could be found.