Genotyping the CRHR1 rs242939 (A>G) Polymorphism by a One-Step Tetra Primer-Amplification Refractory Mutation System-Polymerase Chain Reaction

摘要

Aims: With the rapid advances in molecular techniques, various methods for genotyping single-nucleotide polymorphisms (SNPs) are available. Still, the search for easy, robust, and less costly techniques continues. We wished to develop a Tetra primer-amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) based technique for the corticotrophin-releasing hormone receptor 1 (CRHR1) (rs242939) SNP for use in our research lab. Methods and Results: To detect SNPs in a single-step PCR, we set up two genotyping methods, T-ARMS-PCR and restriction fragment length polymorphism (RFLP). The study was performed using thirty blood samples taken from clinically defined asthmatic patients. The efficiency and effectiveness of results obtained by T-ARMS were validated by PCR-RFLP and sequencing. This study demonstrates that T-ARMS is feasible and applicable to discriminate a wild-type allele from the respective mutant allele in one step. Conclusions: This work is the first that presents a rapid, sensitive, and high throughput genotyping method for the CRHR1 (rs242939) polymorphism and can be used for both large- and small-scale genotyping studies.