基于引物延伸反应进行SNP基因分型的电化学方法

Electrochemical Detection for SNP Genotyping Based on Primer Extension Reaction

引物延伸反应的高特异性使其成为单核苷酸多态性(SNP)基因分型的最常用方法.本文利用引物延伸反应,通过二茂铁标记的dUTP将二茂铁引入到延伸的产物中,用一条捕获探针将延伸产物捕获到电极表面,用差分脉冲伏安法对电极表面的二茂铁进行检测,从而实现了SNP基因分型.考察了延伸反应的退火温度、聚合酶用量以及DNA杂交温度等因素的影响.应用该方法对β-地中海贫血基因密码子28位单碱基突变进行检测,获得了满意的基因分型结果.该方法检测限可达到0.86fmol/L,是一种简便、快速且灵敏的SNP分型方法.

The primer extension（PEXT） reaction is one of the methods most commonly used in genotyping of single nucleotide polymorphisms （SNPs）. Owing to the high specificity of PEXT, the extension reaction can be performed favorably only when the primer and target DNA are perfectly matched. The ferrocene can be incorporated in the extension product by primer extension reaction, which is then captured by the capture probe self-assembled on the electrode surface. Differential pulse voltammetry（DPV） was used to detect the presence of the ferrocene in close proximity of the gold electrode surface. The effects of the annealing temperature, concentration of polymerse and the hybridization temperature on the peak current were evaluated in detail. Furthermore, this proposed method was successfully applied to the gcnotyping of SNPs at β-Thalassemia（ CD28 ）. The detection limit of this method can be reached down to 0. 86 fmol/L and the experimental results show that it is a simple, speedy and sensitive approach for genotyping of SNPs.