Establishment of an optimized PCR method using sequence-specific primers for screening multiple gene polymorphisms simultaneously

Abstract

The present study aimed to explore the factors that affect polymerase chain reaction using sequence-specific primers (PCR-SSP) and to establish an optimized PCR-SSP method for detecting multiple gene polymorphisms simultaneously. The amplification system parameters, including the concentrations of Mg2+, dNTPs, pfu Taq, primers and control primers, were optimized using the designed PCR-SSP reactions. The resulting optimized reaction system was used to determine the melting temperature of the genomic DNA and the cycling parameters. The optimized PCR-SSP method was used to analyze the polymorphisms of the following genes: mutations -308A/G and -238G/A in TNF-α, -174G/C in IL-6 and C/T mutation at exon 188 of CYP2D6 *10B. The PCR-SSP amplification system was optimized; in a 20µl reaction system, the quantities of Mg2+, dNTPs, pfu Taq, primers, control primers and genomic DNA were 3.25µM, 0.5mM, 2.5units, 0.5µM, 0.2µM and 0.15µg, respectively. The cycling system comprised 5 start cycles and took 15min to melt a genomic DNA sample using a touchdown protocol. The optimized PCR-SSP is suitable for polymorphism analysis of polygenic SNPs in large genomic DNA samples and a number of different genes.