A Mutagenic Primer Assay for Genotyping of theCRHR1Gene Rare Variant rs1876828 (A/G) in Asians: A Cost-Effective SNP Typing

Abstract

Background: Today, the genetic and genomic research entered in a new era of high-throughput genotyping technology. However, mutagenic polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is still a choice of genotyping method in molecular epidemiological research. It has been extensively used for the detection of risk alleles, if the target SNP has no natural discriminating restriction site. We undertook this study to develop a mutagenic primer assay for a CRHR1 rare gene variant: rs1876828 (A/G) and to determine their allele frequency in north Indian children. Methods: The mutagenic primers were designed and assay conditions were optimized to perform mutagenic PCR-RFLP in 550 subjects. The efficiency of assay and results were validated by sequencing. Results: This study demonstrated that the mutagenic primer assay is feasible and applicable to discriminate CRHR1 gene rare variant rs1876828 (A/G) and the "frequency of allele "G" was 100% in north Indian asthmatics as well as normal subjects. Conclusion: This method can be used for both large- and small-scale study of complex genetic, where CRHR1 gene plays the pivotal roles.