Amplification of human VKOR gene using PCR for use in cloning & expression / Nurul Ashikin Mustafa

Mutations of human VKOR gene leading to warfarin resistance in certain patients and polymorphism which is associated with a significantly higher risk of aortic calcification in certain patients have been identified. By identification of VKOR gene defects, prevention or treatment inititative such as gene therapy may be initiated for affected patients. PCR is a technique to amplify specific DNA sequences in vitro. PCR is able to rapidly amplify a specific region of a single DNA molecule to yield sufficient quantities that can be cloned, sequenced, or analysed by sequences mapping . The aim of this study is to amplify human VKOR gene using PCR for use in cloning and expression. In this study, specific primers flanking the complete VKOR coding region was designed. The specificity of the primers were evaluated using Oligo Explorer 1.2 Software. The PCR was then performed to detect the VKOR gene. The PCR conditions were optimized by varying the PCR temperature and time. In order to prove the target PCR products were obtained after amplification, the PCR products were separated by gel electrophoresis and visualized under UV transilluminator. The band of interest which was 492 base pairs in size were obtained after human VKOR gene amplification was performed. The amplicon can then be used for cloning and expression of YKOR enzyme. However, more studies need to be performed for more specific amplification without non-specific co-amplification.