AS-PCR检测ABCG2 34G>A多态性

Genotyping of ABCG2 34G>A polymorphism by allele-specific PCR

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。

AIM. To establish an allele-specif- ic PCR method to genotype ABCG2 34 G〉 A polymorphism and verify it using 100 healthy subjects gDNA samples. METHODS： Allele- specific PCR primers with or without an addi- tional mismatch nucleotide artificially introduced within the two bases closest to the 3＇ end were designed, and with different primer pairs, AS- PCR was used to detect ABCG2 34 allele of 100 gDNA samples. The Genotyping results were compared to pyrosequencing results of the same samples we did before. RESULTS：The discrimi- nation ability of 34F/34Rn2 primer pairs was significantly better than the others, and the fre- quency of ABCG2 34AA, 34GA and 34GG of the 100 gDNA samples were 7% ,32% and 61% re- spectively, which was consistent with pyrose- quencing. CONCLUSION. The results suggest the discrimination ability of AS-PCR method es- tablished in this study has been greatly improved and that will facilitate this time-saving, rapid and inexpensive technique into clinical applica- tion.