Optimal Temperature Selection for Mutation Detection by Denaturing HPLC and Comparison to Single-Stranded Conformation Polymorphism and Heteroduplex Analysis
作者： Alistair C Jones , Jehannine Austin , Nancy Hansen , Bastiaan Hoogendoorn , Peter J Oefner
DOI: 10.1093/CLINCHEM/45.8.1133
关键词: DNA 、 Single-strand conformation polymorphism 、 Denaturing high performance liquid chromatography 、 Genetics 、 Biology 、 Mutation detection 、 Molecular biology 、 DNA sequencing 、 Heteroduplex 、 Polymerase chain reaction 、 Denaturing hplc
摘要: Background: Denaturing HPLC (DHPLC) is a semi-automated method for detecting unknown DNA sequence variants. The sensitivity of the dependent on temperature at which analysis undertaken, selection operator experience. To circumvent this, software has been developed predicting optimal DHPLC analysis. We examined utility this software. Methods: maximize relevance our data other investigators, we have screened 42 different amplimers from CFTR, TSC1, and TSC2. samples consisted 103 unique heterozygotes 126 wild-type homozygous controls. Results: At recommended by software, 96% (99 103) all controls were correctly classified. This compares favorably with sensitivities 85% single-stranded conformation polymorphism 82% gel-based heteroduplex analyses same fragments. Conclusions: Software-optimized highly sensitive mutation detection. However, where >96% required, suggest that in addition to temperature, fragments should also be run plus 2 °C.
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