A novel multiplex PCR-RFLP method for simultaneous detection of the MTHFR 677 C %26gt; T, eNOS +894 G %26gt; T and - eNOS -786 T %26gt; C variants among Malaysian Malays

作者：Loo Keat Wei^*; Griffiths Lyn Robyn; Gan Siew Hua

来源：BMC Medical Genetics, 2012, 13: 34.

DOI：10.1186/1471-2350-13-34

Background: Hyperhomocysteinemia as a consequence of the MTHFR 677 C %26gt; 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 %26gt; T and eNOS -786 T %26gt; 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 %26gt; T and eNOS +894 G %26gt; T and eNOS -786 T %26gt; C variants was developed. A total of 114 healthy Malay subjects were recruited. The MTHFR 677 C %26gt; T and eNOS +894 G %26gt; T and eNOS -786 T %26gt; 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 %26gt; T and eNOS +894 G %26gt; T and eNOS -786 T %26gt; C were calculated using the Hardy Weinberg equation. %26lt;br%26gt;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. %26lt;br%26gt;Results: The allele frequencies for MTHFR 677 C %26gt; T were 0.89 (C allele) and 0.11 (T allele); for eNOS +894 G %26gt; T, the allele frequencies were 0.58 (G allele) and 0.43 (T allele); and for eNOS -786 T %26gt; C, the allele frequencies were 0.87 (T allele) and 0.13 (C allele). %26lt;br%26gt;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 %26gt; T and eNOS +894 G %26gt; T and eNOS -786 T %26gt; C variants simultaneously with 100% concordance from DNA sequencing data. This method can be routinely used for rapid investigation of the MTHFR 677 C %26gt; T and eNOS +894 G %26gt; T and eNOS -786 T %26gt; C variants.

• 出版日期2012-5-17