Development of an Allele Specific PCR Method for the Genotyping of CYP2C9 Mutations Isolated from Oral Fluid Samples.

摘要

Warfarin is the most commonly prescribed anticoagulant worldwide, but is also notorious for its narrow therapeutic range, resulting in extreme sensitivity among the population. To more precisely control dosing, two genes have been identified that are highly associated with individual sensitivities to warfarin. One gene is the CYP2C9 gene involved in metabolism of warfarin, having two alleles important to sensitivity: CYP2C9*2 and CYP2C9*3. Both alleles arise from a single nucleotide polymorphism (SNP) that occurs at two separate regions of the gene. The presence of either of these alleles affects the metabolism of the drug requiring a lower than normal dose of warfarin to avoid adverse side effects. Thus, pharmacogenomics have emerged as an important part of the warfarin dosing process.;In the study presented, an assay was developed for selective PCR amplification of the targeted alleles in the CYP2C9 gene important to warfarin sensitivity following DNA extraction from oral fluid samples. A DNA extraction method was developed, and was shown to extract DNA suitable for PCR amplification from oral fluids. Special locked nucleic acid (LNA) primers were designed to be complimentary to both the wild type and mutated alleles in both the CYP2C9*2 and CYP2C9*3 regions. The appropriate conditions at which the primers would selectively amplify only the allele fully complementary to the primer were determined based upon annealing temperature selectivity. The optimized conditions were proven to use an annealing temperature of 67.6°C, at which the wild type and mutated alleles of both CYP2C9*2 and CYP2C9*3 regions could be selectively amplified and genotyped.