Sharma Neeraj, Awasthi Shally, Phadke Subha R, Gupta Sarika
Translational Medicine Unit, Department of Paediatrics, CSMMU, Lucknow, India.
Genet Test Mol Biomarkers. 2012 Jul;16(7):794-7. doi: 10.1089/gtmb.2011.0263. Epub 2012 Feb 21.
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.
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.
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.
随着分子技术的迅速发展，有多种对单核苷酸多态性（SNP）进行基因分型的方法。然而，人们仍在寻找简便、可靠且成本较低的技术。我们希望开发一种基于四引物扩增阻滞突变系统-聚合酶链反应（T-ARMS-PCR）的技术，用于在我们的研究实验室中对促肾上腺皮质激素释放激素受体1（CRHR1）（rs242939）的SNP进行基因分型。
为了在一步PCR中检测SNP，我们建立了两种基因分型方法，即T-ARMS-PCR和限制性片段长度多态性（RFLP）。该研究使用了从临床确诊的哮喘患者中采集的30份血样。通过PCR-RFLP和测序验证了T-ARMS获得的结果的效率和有效性。本研究表明，T-ARMS是可行的，并且可用于一步区分野生型等位基因和各自的突变等位基因。
本研究首次提出了一种针对CRHR1（rs242939）多态性的快速、灵敏且高通量的基因分型方法，可用于大规模和小规模的基因分型研究。
