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

Loading...
Thumbnail Image
File version
Author(s)
Loo, Keat Wei
Griffiths, Lyn
Gan, Siew Hua
Griffith University Author(s)
Primary Supervisor
Other Supervisors
Editor(s)
Date
2012
Size

806406 bytes

File type(s)

application/pdf

Location
Abstract

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.

Journal Title

BMC Medical Genetics

Conference Title
Book Title
Edition
Volume

13

Issue
Thesis Type
Degree Program
School
Publisher link
Patent number
Funder(s)
Grant identifier(s)
Rights Statement
Rights Statement

© 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.

Item Access Status
Note

Page numbers are not for citation purposes. Instead, this article has the unique article number of 34.

Access the data
Related item(s)
Subject

Medical and Health Sciences not elsewhere classified

Genetics

Clinical Sciences

Persistent link to this record
Citation
Collections