Development of the multiplex system for determining markers of predisposition to cardiovascular diseases

Abstract

A multiplex system to detect mutations at loci rs5985, rs1799983, rs5918, rs2243093, rs4673, rs4646994, rs1722009, rs3980933, rs71103505 associated with the development of cardiovascular diseases has been developed. These mutations belong to different types - SNP, STR, Ins/Del - therefore, minisequencing and fragment analysis technologies were used to determine them. Oligonucleotide analysis was performed to amplify all loci in a single reaction format. The minisequencing technology, in comparison with fragment analysis, required additional stages of sample preparation; therefore, oligonucleotides for loci with SNP were combined into a separate plex. For the two plexes created, the same composition of the amplification mixture and parameters of the PCR reaction program were optimized, and “bin” panels were developed to interpret the results. The testing of the system confirmed the possibility of detecting mutations at nine loci with high sensitivity and reproducibility.