Simple Method to Genotype the ACTN3 r577x Polymorphism

Abstract

The alpha-actinin-3 r577x polymorphism (rs1815739) is one of the most important polymorphisms associated with athletic performance. This single-nucleotide mutation leads to a premature stop codon, resulting in a nonfunctional protein product. The presence of the dominant R allele is associated with full power skeletal muscle contraction. Homozygosity for the X allele is correlated with more efficient energy disposure. Restriction fragment length polymorphism and real-time polymerase chain reaction (PCR) are the standard methods used to genotype this polymorphism, but they are expensive and require special equipments. Here, we present a simple and cost-efficient method to genotype the ACTN3 r577x polymorphism by a single PCR. External primers yield a 690-bp product that indicates the template quality. Internal primers produce a 413-bp product if the R allele is present and a 318-bp product if the X allele is present. Our four-primer genotyping PCR was validated by the standard real-time PCR, generally used to genotype this single-nucleotide polymorphism, demonstrating the accuracy of this method. This protocol is perfect for small- or large-scale cohort genotyping of the ACTN3 r577x polymorphism.

The ACTN3 R577X Polymorphism Is Associated with Cardiometabolic Fitness in Healthy Young Adults

“…Finally, we used serum metabolomics to identify for the first time, a role for the ACTN3 R577X polymorphism in human lipid metabolism. Taken together, these associations suggest that prospective and retrospective ACTN3 genotyping [ 48 ] may provide novel insights into human athletic performance and cardiovascular disease risk stratification.…”

Association between ACTN3 and acute mountain sickness

“…The technique allows the analysis of the two variant alleles of a SNP in a particular segment of DNA. For this experimental approach, TaqMan® assays that have already been functionally tested ( ACTN3 rs1815729) were used, because they offer better performance and more affordable cost compared to the customization of the tests [29].…”

Genetic Variants and Mixed Sport Disciplines: A Comparison among Soccer, Combat and Motorcycle Athletes

“…Determination of ACE, ACTN3, PPARα, and CK-MM, genotypes. Genotyping of the ACE (rs4646994) and ACTN3 (rs1815739) polymorphisms were assessed by polymerase chain reaction (PCR) using primers previously described (23,24). Amplification of ACE was performed in a final volume of 25 µl containing 2 μl genomic DNA template, 12.5 μl of 2X PCR Master Mix (Fermentas, ThermoFisher Scientific), 1 µl of each primer (10 µM; Eurofins MWG Operon) and 8.5 μl DEPC-treated water.…”

“…(2015) (24). ACTN3 gene PCR amplification was performed as follows: 95 °C for 5 min, followed by 35 cycles at 95 °C for 20 sec, 67 °C for 20 sec and at 72 °C for 45 sec, followed by a final step at 72 °C for 3 min.…”