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目的:建立和优化检测ABCG2 34G>A多态性的等位基因特异性PCR方法(allele-specific PCR,AS-PCR),并对100例健康受试者进行分型检测。方法:设计合成3'-末端错配引物和包含内部错配位点的3'-末端错配引物,进行AS-PCR扩增,比较两者的扩增特异性,应用AS-PCR检测100例健康受试者ABCG2 34位多态型,采用焦磷酸测序方法(pyrosequencing)进行抽样验证。结果:含有第3位内部错配位点的3'-末端错配引物扩增特异性明显优于含有第2位内部错配位点以及不含内部错配位点的3'-末端错配引物。100例样本AS-PCR分型结果与同批标本前期的焦磷酸测序结果一致。ABCG2 34AA,34GA和34GG型频率分别为7%、32%、61%。结论:本文所建立的AS-PCR方法在进行ABCG2 34位多态分型时,具有省时、快速和成本低等优点,经过在引物中引入内部错配位点等优化设计后,分型结果准确可靠,适合临床应用。
OBJECTIVE: To establish and optimize allele-specific PCR (AS-PCR) for detecting ABCG2 34G> A polymorphism and to detect 100 healthy subjects. Methods: The 3'-end mismatch primer and the 3'-end mismatch primer containing the internal mismatch site were designed and synthesized. The AS-PCR amplification was performed. The amplification specificity of the two primers was compared. 100 samples were detected by AS-PCR Healthy subject ABCG2 34 polymorphism, pyrosequencing method (pyrosequencing) sampling verification. Results: The amplification specificity of the 3'-end mismatch primer containing the third internal mismatch site was significantly better than that of the 3'-end mismatch site containing the second internal mismatch site and no internal mismatch site Primer. 100 samples AS-PCR typing results with the same batch of pre-pyrosequencing results consistent. ABCG2 34AA, 34GA and 34GG frequency were 7%, 32%, 61%. Conclusion: The AS-PCR method established in this paper has the advantages of time saving, fastness and low cost when performing ABCG2 34 polymorphism typing. After the optimal design of introducing internal mismatch sites into the primers, the typing results Accurate and reliable, suitable for clinical application.