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Vitamin K antagonists are anticoagulants which represent widely prescribed drugs for prevention and treatment of thromboembolic disorders. The molecular target of these drugs is vitamin K epoxide reductase enzyme and their metabolism is performed by the cytochrome P-450 2C9 enzyme, both of which are encoded by polymorphic genes - vitamin K epoxide reductase complex subunit 1 (VKORC1) and cytochrome P-450 2C9 (CYP2C9), respectively. Identification of VKORC1 and CYP2C9 genotypes was recommended by the U.S. Food and Drug Administration in 2007, in order to guide the initial dosing of warfarin, the first oral vitamin K antagonist drug, to achieve optimum anticoagulation and prevent hemorrhagic events. This protocol is designed for undergraduate students of pharmacology or medicine, or anyone receiving training in pharmacogenomics/pharmacogenetics and understanding a relationship between a gene and drug response for the first time. The protocol represents a step-by-step guide to using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method in determining VKORC1 -1639G>A, CYP2C9*1/*1, CYP2C9*1/*2, CYP2C9*1/*3, and CYP2C9*2/*3 genotypes. Based on the combination of the VKORC1 -1639G>A and CYP2C9 genotypes, that is, the pattern of DNA fragments, which are visualized using agarose gel electrophoresis and DNA staining, a conclusion on an individual’s sensitivity to vitamin K antagonists and appropriate drug dose is reached. |
