Two-dye based arrayed primer extension for simultaneous multigene detection in lipid metabolism.

Cardiovascular disease (CVD) is one of the major causes of death worldwide. Numerous genetic risk factors in lipid metabolism, including mutations of LDLR, APOB, and PCSK9, as well as polymorphisms of CETP and APOE, have been found to associate with CVD. In this study, a two-dye based arrayed primer extension (APEX) microarray assay for simultaneous multigene (LDLR, APOB, PCSK9, CETP, and APOE) detection was developed. The DNA templates, originating from 1 DNA sample of known genotype and 7 blind DNA samples, were amplified by uniplex PCR. Optimized conditions for the APEX reaction were determined to include a hybridization temperature of 55°C and a DNA template size of 50-150bp. The total assay including PCR, purification, fragmentation, APEX reaction, and image analysis could be performed in 6h. In total, 48 genotypes were identified among 8 individual DNA samples by APEX analysis. The data suggest that this APEX microarray offers a robust, fast, and versatile option for screening these genotypes in hypercholesterolemia patients.