A novel multiplex PCR-RFLP method for simultaneous detection of the MTHFR 677 C>T, eNOS +894 G>T and - eNOS -786 T>C variants among Malaysian Malays
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BACKGROUND: 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 eNOS +894 G?>?T and eNOS -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 eNOS +894 G?>?T and eNOS -786 T?>?C variants was developed. A total of 114 healthy Malay subjects were recruited. The MTHFR 677 C?>?T and eNOS +894 G?>?T and eNOS -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 eNOS +894 G?>?T and eNOS -786 T?>?C were calculated using the Hardy Weinberg equation. METHODS: 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. RESULTS: The allele frequencies for MTHFR 677 C?>?T were 0.89 (C allele) and 0.11 (T allele); for eNOS +894 G?>?T, the allele frequencies were 0.58 (G allele) and 0.43 (T allele); and for eNOS -786 T?>?C, the allele frequencies were 0.87 (T allele) and 0.13 (C allele). CONCLUSIONS: Our PCR-RFLP method is a simple, cost-effective and time-saving method. It can be used to successfully genotype subjects for the MTHFR 677 C?>?T and eNOS +894 G?>?T and eNOS -786 T?>?C variants simultaneously with 100% concordance from DNA sequencing data. This method can be routinely used for rapid investigation of the MTHFR 677 C?>?T and eNOS +894 G?>?T and eNOS -786 T?>?C variants.
BMC Medical Genetics
© 2012 Loo et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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