Background: Recently, Denaturing High-Performance Liquid Chromatography (DHPLC) has been widely used for mutation detection hMLH1 and hMSH2 genes due to reduced cost of analysis, accuracy, and high sample throughput. Unfortunately, one major drawback in screening the hMLH1 and hMSH2 genes with any analysis technique involves sample preparation. Additionally, there are limitations to this technique which include: (1) amplicons for hMLH1 and hMSH2 exons cannot be generated under the same PCR thermal cycler condition due to differences in the annealing temperatures of the traditional primer sets which drastically increases sample preparation time; (2) due to minimal changes in the DHPLC chromatogram when compared to the corresponding wild-type amplicon, there is a possibility to not detect a homozygous mutation; and (3) lack of specialized mutation analysis software for automated screening of the hMLH1 and hMSH2 genes with the Transgenomic Wave system.
Methods: To overcome these limitations, the hMLH1 and hMSH2 condition-oriented-PCR primer-embedded-reactor (COPPER) plate was developed to reduce the sample preparation time and technological skill required for analysis, as well as standardize a mutation screening technique for hMLH1 and hMSH2 analysis using the Transgenomic Wave system for research and clinical genetics investigations. In this study, we validated the COPPER plate for simultaneous amplification of the exons of the hMLH1 and hMSH2 genes coupled to DHPLC detection for colorectal cancer (CRC) patients.
Results and conclusions: Our results suggest that the COPPER plate DHPLC approach is a simple, cost effective, accurate, universal, and reproducible technology for screening hMLH1 and hMSH2 genes which are associated with human CRC. We also believe that the COPPER plate DHPLC approach is amenable for characterization of other germline alterations in clinical genetics and pharmacogenetics.