Electrochemical Detection of SNP in Human Mitochondrial DNA Using Cyclic Primer Extension with Biotinylated Nucletides and Enzymatic Labeling at Disposable Pencil Graphite Electrodes,Electroanalysis

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Electrochemical Detection of SNP in Human Mitochondrial DNA Using Cyclic Primer Extension with Biotinylated Nucletides and Enzymatic Labeling at Disposable Pencil Graphite Electrodes
Electroanalysis ( IF 2.7 ) Pub Date : 2018-07-16 , DOI: 10.1002/elan.201800314
Medard Plucnara ₁ , Ece Eksin _{2,

3} , Arzum Erdem _{2,

3} , Miroslav Fojta ₁

Affiliation

A novel method of SNP typing in human mitochondrial DNA utilizing enzymatic labeling and electrochemical detection at disposable pencil graphite electrodes is described. The procedure is based on amplification of DNA stretches by cyclic primer extension (PEx) of SNP‐specific diagnostic primers in a mixture of biotinylated and natural nucleotides. The diagnostic primers are designed to recognize, by its 3'‐terminal nucleotide, the SNP‐site in target template. Under optimized conditions of the PEx reaction, efficient polymerase synthesis of biotin‐labeled strands takes place only in the case of full complementarity between the diagnostic primer and the target SNP site. There is also benefit from introducing many biotin molecules per extended DNA strand, resulting in another level of signal amplification. After adsorption of biotinylated PEx products at the electrode surface, streptavidin‐alkaline phosphatase conjugate was bound to the biotin tags, 1‐naphthol was enzymatically produced and electrochemically detected. Several critical steps and parameters of the assay, including termination of 3’‐OH ends of residual amplification primers, temperature for annealing of diagnostic primers, relative amount of biotinylated deoxynucleoside triphosphate in the PEx mixture and number of PEx cycles were optimized in this study to attain best SNP resolution, and reduction of time needed for the analysis.

中文翻译：

电化学检测人线粒体DNA中的SNP，使用生物素化的核苷酸循环引物延伸并在一次性铅笔状石墨电极上进行酶标记。

描述了一种利用酶标记和在一次性铅笔石墨电极上进行电化学检测在人线粒体DNA中进行SNP分型的新方法。该程序基于生物素化和天然核苷酸混合物中SNP特异性诊断引物的环状引物延伸（PEx）对DNA片段的扩增。诊断引物旨在通过其3'端核苷酸识别靶模板中的SNP位点。在PEx反应的最佳条件下，只有在诊断引物和目标SNP位点完全互补的情况下，生物素标记的链才可以进行有效的聚合酶合成。每条延伸的DNA链引入许多生物素分子，也会带来另一级信号放大，这也带来了好处。将生物素化的PEx产物吸附在电极表面后，将链霉亲和素碱性磷酸酶偶联物结合到生物素标签上，酶促生成1-萘酚并进行电化学检测。在这项研究中，优化了测定的几个关键步骤和参数，包括终止残留扩增引物的3'-OH末端，诊断引物退火的温度，PEX混合物中生物素化的脱氧核苷三磷酸的相对量以及PEx循环数，获得最佳的SNP分辨率，并减少分析所需的时间。

更新日期：2018-07-16

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