A novel multiplex PCR-RFLP method for simultaneous detection of the <it>MTHFR 677 C > T</it>, <it>eNOS +894 G > T</it> and <it>- eNOS -786 T > C</it> variants among Malaysian Malays

<p>Abstract</p> <p>Background</p> <p>Hyperhomocysteinemia as a consequence of the MTHFR 677 C > T variant is associated with cardiovascular disease and stroke. Another factor that can potentially contribute to these disorders is a depleted nitric oxide level, which can be due to the presence of <it>eNOS</it> +894 G > T and <it>eNOS −</it>786 T > C variants that make an individual more susceptible to endothelial dysfunction. A number of genotyping methods have been developed to investigate these variants. However, simultaneous detection methods using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis are still lacking. In this study, a novel multiplex PCR-RFLP method for the simultaneous detection of MTHFR 677 C > T and <it>eNOS</it> +894 G > T and <it>eNOS −</it>786 T > C variants was developed. A total of 114 healthy Malay subjects were recruited. The MTHFR 677 C > T and <it>eNOS</it> +894 G > T and <it>eNOS −</it>786 T > C variants were genotyped using the novel multiplex PCR-RFLP and confirmed by DNA sequencing as well as snpBLAST. Allele frequencies of MTHFR 677 C > T and <it>eNOS</it> +894 G > T and <it>eNOS −</it>786 T > C were calculated using the Hardy Weinberg equation.</p> <p>Methods</p> <p>The 114 healthy volunteers were recruited for this study, and their DNA was extracted. Primer pair was designed using Primer 3 Software version 0.4.0 and validated against the BLAST database. The primer specificity, functionality and annealing temperature were tested using uniplex PCR methods that were later combined into a single multiplex PCR. Restriction Fragment Length Polymorphism (RFLP) was performed in three separate tubes followed by agarose gel electrophoresis. PCR product residual was purified and sent for DNA sequencing.</p> <p>Results</p> <p>The allele frequencies for MTHFR 677 C > T were 0.89 (C allele) and 0.11 (T allele); for <it>eNOS</it> +894 G > T, the allele frequencies were 0.58 (G allele) and 0.43 (T allele); and for <it>eNOS −</it>786 T > C, the allele frequencies were 0.87 (T allele) and 0.13 (C allele).</p> <p>Conclusions</p> <p>Our PCR-RFLP method is a simple, cost-effective and time-saving method. It can be used to successfully genotype subjects for the <it>MTHFR 677 C > T</it> and <it>eNOS +894 G > T</it> and <it>eNOS −786 T > C</it> variants simultaneously with 100% concordance from DNA sequencing data. This method can be routinely used for rapid investigation of the <it>MTHFR 677 C > T</it> and <it>eNOS +894 G > T</it> and <it>eNOS −786 T > C</it> variants.</p