Development of an Allele-specific PCR for Detection of VKORC1 -1639G > A and 1173C > T for Warfarin Therapy
Article Sidebar
Main Article Content
Abstract
Warfarin is an anticoagulant drug, which has a narrow therapeutic index VKORC1 gene
polymorphisms [(-1639G > A (rs9923231) and 1173C > T (rs9934438)] affect drug responses that need to be considered for warfarin dosage requirement. Therefore, the objective of this study was to develop Allelespecific PCR (AS-PCR). Firstly, specific primers for VKORC1 gene were designed. PCR optimization was performed by varying annealing temperature. The results showed that 1.25 mM MgCl2 was suitable for all primers. At -1639G > A, 65๐C was suitable annealing temperature for mutant and control primers. Whereas, 57๐C was suitable annealing temperature for wildtype primers and all primer pairs in another position, 1173C > T. PCR products were validated by DNA sequencing, resulting in 100% homology. In conclusion, the newly developed AS-PCR could use for VKORC1 genotype detection to adjust for warfarin dosage.
Article Details
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
References
Thongarporn, S. (2012). Prevalence of adverse drug reactions and factors affecting bleeding from warfarin usage in southern Thai patients. Master Thesis. Prince of Songkla University.
Charuchotekamol, A. (2010). Warfarin and genetic polymorphism. Thai Pharmaceutical and Health Science Journal, 5(2), 180–184.
Choicharnchaikul, S., Malathum, P., & Karnchanachari, S. (2008). The use of warfarin in older adults: a case study and caring. Ramathibodi Nursing Journal, 14(3), 366–384.
Ansell, J., Hirsh, J., Hylek, E., Jacobson, A., Crowther, M., & Palareti, G. (2008). Pharmacology and management of the vitamin K antagonists: american college of chest physicians evidence-based clinical practice guidelines (8th edition). Chest, 133(6 Suppl), 160S–198S. DOI: 10.1378/chest.08-0670.
Rettie, A.E., & Tai, G. (2006). The pharmacogenomics of warfarin: closing in on personalized medicine. Molecular Interventions, 6(4), 223–227. DOI: 10.1124/mi.6.4.8.
Limdi, N.A., Wadelius, M., Cavallari, L., Eriksson, N., Crawford, D.C., Lee, M.M., …Wagner, (2010). Warfarin pharmacogenetics: a single VKORC1 polymorphism is predictive of dose across 3 racial groups. Blood, 115(18), 3827–3834. DOI: https://doi.org/10.1182/blood- 2009-12-255992.
Sambrook, J.F., & Russell, D.W. (2001). Molecular cloning: A Laboratory Manual. (3rd Ed). New York: Cold Spring Harbor Laboratory Press.
Liu, J., Huang, S., Sun, M., Liu, S., Liu, Y., Wang, W.,... Hua, W. (2012). An improved allele-specific PCR primer design method for SNP marker analysis and its application. Plant Methods, 8, 34 (2012). DOI: https://doi.org/10.1186/1746-4811-8-34.
Park, Y.H., & Kohel, R.J. (1994). Effect of concentration of MgCl2 on random-amplified DNA polymorphism. Biotechniques, 16(4), 652–656.