Label-Free Voltammetric Detection of Products of Terminal Deoxynucleotidyl Transferase Tailing Reaction,Electroanalysis

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Label-Free Voltammetric Detection of Products of Terminal Deoxynucleotidyl Transferase Tailing Reaction
Electroanalysis ( IF 2.7 ) Pub Date : 2018-08-24 , DOI: 10.1002/elan.201800452
Monika Hermanová ₁ , Pavlína Havranová-Vidláková ₁ , Anna Ondráčková ₁ , Swathi Senthil Kumar ₂ , Richard Bowater ₂ , Miroslav Fojta _{1,

3}

Affiliation

A label-free approach that takes advantage of intrinsic electrochemical activity of nucleobases has been applied to study the products of terminal deoxynucleotidyl transferase (TdT) tailing reaction. DNA homooligonucleotides A 30 , C 30 and T 30 were used as primers for the tailing reaction to which a dNTP – or a mixture of dNTPs – and TdT were added to form the tails. Electrochemical detection enabled study of the tailing reaction products created by various combinations of primers and dNTPs, with pyrolytic graphite electrode (PGE) being suitable for remarkably precise analysis of the length of tailing reaction products. Furthermore, the hanging mercury drop electrode (HMDE) was able to reveal formation of various DNA structures, such as DNA hairpins and G-quadruplexes, which influence the behavior of DNA molecules at the negatively charged surface of HMDE. Thus, the described approach proves to be an excellent tool for studying the TdT tailing reactions and for exploring how various DNA structures affect both the tailing reactions and electrochemical behavior of DNA oligonucleotides at electrode surfaces.

中文翻译：

末端脱氧核苷酸转移酶加尾反应产物的无标记伏安法检测

一种利用核碱基固有电化学活性的无标记方法已被应用于研究末端脱氧核苷酸转移酶 (TdT) 拖尾反应的产物。DNA 同源寡核苷酸 A 30 、C 30 和 T 30 用作加尾反应的引物，向其中添加 dNTP（或 dNTP 的混合物）和 TdT 以形成尾。电化学检测能够研究由引物和 dNTP 的各种组合产生的拖尾反应产物，热解石墨电极 (PGE) 适合对拖尾反应产物的长度进行非常精确的分析。此外，悬汞滴电极 (HMDE) 能够揭示各种 DNA 结构的形成，例如 DNA 发夹和 G-四链体，这会影响 HMDE 带负电表面的 DNA 分子的行为。因此，所描述的方法被证明是研究 TdT 拖尾反应和探索各种 DNA 结构如何影响电极表面 DNA 寡核苷酸的拖尾反应和电化学行为的绝佳工具。

更新日期：2018-08-24

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