Typing of Eight CYP450 Loci Based on Multiplex AS-PCR Techniques

Abstract

In the era of personalized medicine, the study of single-nucleotide polymorphisms (SNPs) in cytochrome P450 (CYP450) family genes is becoming increasingly important. The accurate genotyping of SNPs is of great significance for analyzing individual differences in drug metabolism and disease susceptibility, and promoting clinical precision medicine. Therefore, developing an efficient, accurate, and cost-effective detection scheme is extremely necessary. This study combined TaqMan probe technology with allele-specific PCR (AS-PCR), and by using a single-nucleotide ultra-high-fidelity polymerase, successfully developed an efficient and accurate 4-channel multiplex detection protocol for key CYP450 genes. This protocol accurately identified CYP450 gene alleles while eliminating interference from highly homologous sequences, and its specificity and accuracy were verified. The results showed that the cycle threshold differences (ΔC_t values) of different SNP alleles and highly homologous segments in the same detection system could reach more than 4. The detection results were consistent with the sequencing results, and the method could better identify the genotypes of eight SNPs (rs1799853, rs1057910, rs2242480, rs776746, rs2069514, rs2070676, rs6413432, and rs890293). The detection protocol developed in this study offers strong specificity, high accuracy, and simple operation, while achieving a favorable balance among detection speed, cost, and accuracy. It can be readily applied in pharmacogenomics research.

Keywords

multiplex AS-PCR / cytochrome P450 (CYP450) / genotyping / single-nucleotide ultra-high-fidelity polymerase

Funding

National Key R&D Program of China(2018YFA0801101)