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ALLELE-SPECIFIC POLYMERASE CHAIN REACTION (AS-PCR) AS A TOOL FOR GENOTYPING KCNQ1 RS2237892 POLYMORPHISM
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Tóm tắt
Aims: To develop and validate a protocol of allele-specific polymerase chain reaction (AS-PCR) for detecting the KCNQ1 rs2237892 polymorphism.
Methods: Genomic human DNA was extracted from peripheral blood samples of a population-based cohort in Ha Nam province, Vietnam. The AS-PCR protocol was optimized by adjusting annealing temperatures (Ta) and primer concentrations. To ensure accuracy, the genotyping results obtained from AS-PCR were validated against restriction fragment length polymorphism (RFLP) using the PvuII enzyme and direct Sanger sequencing in a subset of samples.
Results: The optimal annealing temperature for the AS-PCR assay was determined to be 59°C, yielding a clear 392-bp diagnostic band. The AS-PCR method demonstrated 100% concordance with both RFLP and Sanger sequencing results across all tested genotypes (CC, CT, and TT). The protocol effectively discriminated between the risk allele (C) and the alternative allele (T) with high sensitivity and specificity.
Conclusion: The established AS-PCR protocol provides a reliable and high-throughput tool for genotyping the KCNQ1 rs2237892 variant. Given its minimal equipment requirements and high accuracy, this method is well-suited for large-scale genetic epidemiological studies and clinical screening in resource-limited settings like Vietnam.
Từ khóa
Type 2 diabetes mellitus, genotyping, rs2237892, KCNQ1 gene, AS-PCR
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Tài liệu tham khảo
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