基于SNaPShot-FCA的乙醛脱氢酶2基因型微量检测

曾树树; 高秀峰; 张又; 冯传芮; 罗亚雄; 吴晓婷; 李永生

基于SNaPShot-FCA的乙醛脱氢酶2基因型微量检测

Microanalysis of Acetaldehyde Dehydrogenase 2 Genotype Based on SNaPShot-FCA

摘要

摘要:
目的建立单碱基末端延伸-荧光毛细分析法(SNaPShot-fluorescence capillary analysis, SNaPShot-FCA)快速检测乙醛脱氢酶2基因(ALDH2)rs671位点的基因型。
方法收集人外周血细胞，提取基因组DNA；采用R6G-ddATP和cy5-ddGTP作为荧光底物，用SNaPShot法扩增ALDH2基因，生成3′末端带有不同荧光标记的DNA产物；琼脂糖凝胶电泳分离并回收产物，用FCA微量检测，根据荧光光谱鉴定ALDH2 rs671的基因型；样本检测均重复3次，并与DNA测序结果相比较。
结果优选的R6G-ddATP和cy5-ddGTP浓度分别为1.4 μmol/L和8.0 μmol/L；在最适条件下运用SNaPShot-FCA对106例样本进行了ALDH2基因型检测，检测结果显示：野生型(GG)67例，杂合型(AG)38例，突变纯合型(AA)1例，与测序结果一致。
结论成功建立了微量检测ALDH2基因型的SNaPShot-FCA，可用于ALDH2基因的快速筛查与鉴定。

Abstract:
Objective To establish SNaPShot-fluorescence capillary analysis (SNaPShot-FCA) assay for rapid detection of the genotype of aldehyde dehydrogenase 2 gene (ALDH2) rs671 locus.
Methods The genomic DNA was extracted from peripheral blood cells. Using R6G-ddATP and cy5-ddGTP as fluorescent substrates, the ALDH2 gene was amplified by SNaPShot to generate DNA products with different fluorescent dyes at the 3′ end. FCA was used to detect the products separated by agarose gel electrophoresis and recovered by gel recovery kit, and the genetype of ALDH2 polymorphism was analyzed by fluorescence spectrum. The samples were tested three times repeatedly and compared with the results of DNA sequencing.
Results The optimal concentrations of R6G-ddATP and cy5-ddGTP were 1.4 μmol/L and 8.0 μmol/L, respectively. 106 samples were tested for ALDH2 genotype by SNaPShot-FCA under optimal conditions, including 67 of wild type (GG), 38 of hybrid type (AG), and 1 of mutant type (AA), which were consistent with the sequencing results.
Conclusion This study successfully established the SNaPShot-FCA for the micro-detection of ALDH2 genotype for the rapid screening and identification of ALDH2 gene.

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