Optimization of fast and simple real-time PCR-based method for genotyping the angiotensin converting enzyme-I I/D polymorphism

Abstract

The insertion or deletion of 288 bp in intron 16 of the gene encoding for angiotensin converting enzyme-I (ACE) was the first genetic variant associated with physical performance and one of the most studied in the past 15 years. Carries of the deletion in one of its alleles may have higher enzyme activity, which may result in a greater vasoconstrictor response. These individuals may also better respond to strength and power training, as carriers of the insertion may have a greater propensity to respond better to the endurance training. Traditionally, to determine an individual genotype (I/I, I/D or D/D) the conventional PCR is the method used. This method involves the PCR reaction and then gel electrophoresis for the visualization of DNA bands indicating the genotype. For use this methodology on a large scale, as seen in association studies, such as those used to evaluate the influence of genetics in sport, this double process of conventional PCR is not time-effective. This paper aims to present an optimized, fast and efficient method for the genotyping of this polymorphism by real-time PCR, using genomic DNA samples collected from buccal cells. The method discussed in the text was originally proposed in 2001, but its original configuration has limitations in its methodology and uses much of the reagent. Thus, protocol variables such as primer concentration, reaction volume and the resolution of dissociation curve which indicates the genotypes were adjusted. After this adjustments the protocol remained effective with a reduced cost, suitable for use in large-scale studies involving genetic and sport.