DEVELOPMENT OF NEW AS-PCR BASED ANALYTICAL APPROACH FOR DETECTING THE SINGLE NUCLEOTIDE POLYMORPHISM OF AGTR.1 GENE

Objective: Angiotensin II is a potent vasoactive peptide that causes blood vessels to constrict, resulting in increased blood pressure. It acts through at least two types of receptors. The angiotensin II receptor type 1 gene (AGTR1) encodes the type 1 receptor (AT1). The AT1 receptor mediates the major cardiovascular effects of angiotensin II. A single nucleotide polymorphism (SNP) in the 3'-untranslated region of the AGTR1 gene (A1166C) has been linked in several studies with essential hypertension. A new analytical approach based on allele-specific polymerase chain reaction (AS-PCR) was developed in this study to detect this SNP.Methods: Allele-specific primers were designed by using appropriate software to allow the PCR amplification only if the nucleotide at the 3'-end of the primer complements the base of the wild-type or variant-type DNA sample. The primers were then tested for uniqueness using the Basic Local Alignment Search Tool search engine. The developed method was tested on 21 samples.Results: The developed method accurately detected the genotype of the SNP. Our results were validated using the reference method for detecting A1166C SNP, PCR-restriction fragment length polymorphism (PCR-RFLP). The use of AS-PCR technique reduced both time and cost of the A1166C AGTR1 genotyping. Moreover, the AS-PCR test is more suitable as it reduces the false results due to incompletely digested PCR products, which can be a problem with PCR-RFLP technique. Conclusion: The use of this method will enable researchers to carry out genetic polymorphism studies for the association of A1166C SNP in AGTR1 gene with essential hypertension and other heart diseases without the use of expensive instrumentation and reagents.Keywords: Essential hypertension, SNP, Angiotensin II type 1 receptor gene (AGTR1), Renin-angiotensin system, AS-PC