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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

DOI: 10.1186/1471-2350-13-34

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Abstract:

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.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).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.

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