Optimal temperature selection for mutation detection by denaturing HPLC and comparison to single-stranded conformation polymorphism and heteroduplex analysis

Background: Denaturing HPLC (DHPLC) is a semiautomated method for detecting unknown DNA sequence variants. The sensitivity of the method is dependent on the temperature at which the analysis is undertaken, the selection of wh ich is dependent on operator experience. To circumvent this, software has b een developed for predicting the optimal temperature for DHPLC analysis. We examined the utility of this software. Methods: To maximize the relevance of our data for other investigators, we have screened 42 different amplimers from CFTR, TSC1, and TSC2. The samples consisted of 103 unique sequence heterozygotes and 126 wild-type homozygou s controls. Results: At the temperature recommended by the software, 96% (99 of 103) of heterozygotes and all of the wild-type controls were correctly classified. This compares favorably with sensitivities of 85% for single-stranded conf ormation polymorphism and 82% for gel-based heteroduplex analyses of the sa me fragments. Conclusions: Software-optimized DHPLC is a highly sensitive method for muta tion detection. However, where sensitivity >96% is required, our data sugge st that in addition to the recommended temperature, fragments should also b e run at the recommended temperature plus 2 degrees C. (C) 1999 American As sociation for Clinical Chemistry.