Table of Contents Author Guidelines Submit a Manuscript
BioMed Research International
Volume 2014, Article ID 316310, 6 pages
http://dx.doi.org/10.1155/2014/316310
Research Article

A New Nested Allele-Specific Multiplex Polymerase Chain Reaction Method for Haplotyping of VKORC1 Gene to Predict Warfarin Sensitivity

1Department of Pharmacology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia
2Department of Haematology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia
3Invasive Cardiology Laboratory, Hospital Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia
4Human Genome Center, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia

Received 26 December 2013; Revised 23 February 2014; Accepted 25 February 2014; Published 30 March 2014

Academic Editor: Stelvio M. Bandiera

Copyright © 2014 Yung An Chua et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The vitamin K epoxide reductase complex 1 gene (VKORC1) is commonly assessed to predict warfarin sensitivity. In this study, a new nested allele-specific multiplex polymerase chain reaction (PCR) method that can simultaneously identify single nucleotide polymorphisms (SNPs) at VKORC1 381, 861, 5808, and 9041 for haplotype analysis was developed and validated. Extracted DNA was amplified in the first PCR DNA, which was optimized by investigating the effects of varying the primer concentrations, annealing temperature, magnesium chloride concentration, enzyme concentration, and the amount of DNA template. The amplification products produced from the first round of PCR were used as templates for a second PCR amplification in which both mutant and wild-type primers were added in separate PCR tubes, followed by optimization in a similar manner. The final PCR products were resolved by agarose gel electrophoresis and further analysed by using a VKORC1 genealogic tree to infer patient haplotypes. Fifty patients were identified to have H1H1, one had H1H2, one had H1H7, 31 had either H1H7 or H1H9, one had H1H9, eight had H7H7, and one had H8H9 haplotypes. This is the first method that is able to infer VKORC1 haplotypes using only conventional PCR methods.