Chua Yung An, Abdullah Wan Zaidah, Yusof Zukurnai, Gan Siew Hua
Department of Pharmacology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.
Department of Haematology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.
Biomed Res Int. 2014;2014:316310. doi: 10.1155/2014/316310. Epub 2014 Mar 30.
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.
通常会评估维生素K环氧化物还原酶复合体1基因（VKORC1）以预测华法林敏感性。在本研究中，开发并验证了一种新的巢式等位基因特异性多重聚合酶链反应（PCR）方法，该方法能够同时鉴定VKORC1基因381、861、5808和9041位点的单核苷酸多态性（SNP）以进行单倍型分析。提取的DNA在第一轮PCR中进行扩增，通过研究改变引物浓度、退火温度、氯化镁浓度、酶浓度和DNA模板量的影响对其进行优化。第一轮PCR产生的扩增产物用作第二轮PCR扩增的模板，在第二轮PCR扩增中，突变型和野生型引物分别添加到不同的PCR管中，然后以类似方式进行优化。最终的PCR产物通过琼脂糖凝胶电泳进行分离，并通过使用VKORC1系谱树进一步分析以推断患者的单倍型。鉴定出50例患者具有H1H1单倍型，1例具有H1H2单倍型，1例具有H1H7单倍型，31例具有H1H7或H1H9单倍型，1例具有H1H9单倍型，8例具有H7H7单倍型，1例具有H8H9单倍型。这是第一种仅使用常规PCR方法就能推断VKORC1单倍型的方法。
