Modified method for analysis of InDel ACE1 gene polymorphism using the temperature melting curves of PCR products

Aim.The ACE1 gene is a key to the renin-angiotensin system, as it encode the ACE1 enzyme, which regulates blood pressure and water-electrolyte balance.The InDel (I/D) polymorphism of the ACE1 gene causes a different level of enzyme synthesis and is associated with various pathological conditions, including the course of COVID-19.The aim of our study was to develop an efficient method for the analysis of I/D polymorphism of the ACE1 gene by analyzing the melting temperature curves of specific PCR products.Methods.Genotyping was carried out by the PCR method using DNA isolated from peripheral blood leukocytes.The primers were designed using BLAST SEARCH and synthesized at METABION (Germany).The reaction was carried out using the "HOT FIREPol EvaGreen qPCR Mix Plus" mixture.Amplification was performed on iQ5TM Multicolor Real-Time PCR Detection System (BIO-RAD), melting curves were analyzed using BIO-RAD iQ5 Optical System Software V 2.0.Results.The proposed method, using one pair of primers, turned out to be effective for detecting genotypes based on the melting temperature curves of PCR products: the peak at 83-85 C corresponded to deletion, at 86-89 C to insertion, and in the presence of both peaks, the genotype was defined as heterozygous.Conclusions.The proposed method of genotyping the I/D polymorphism of the ACE1 gene is simple and reliable, and can be used for the analysis of genetic markers of cardiovascular pathology and the prognosis of the course of COVID-19.K e y w o r d s: 1, InDel, melting temperature curve analysis.