Efficiency and specificity of microRNA-primed nucleotide analog incorporation by various DNA polymerases.
Efficiency and specificity of microRNA-primed nucleotide analog incorporation by various DNA polymerases.
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DOI：
10.1016/j.ab.2009.05.010
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发表时间：
2009-08-15
影响因子：
2.9
通讯作者：
Golovlev V
中科院分区：
文献类型：
--
作者：
Sun Y;Gregory KJ;Golovlev V
microRNA作为基因表达的内源性调控因子，其定量和表达分析引起了人们极大的兴趣。高灵敏度和特异性的microRNA检测技术，如实时PCR和最近引入的生物发光microRNA检测，需要系统地研究用于microRNA的DNA聚合酶。在这项研究中，各种DNA聚合酶已被研究，以评估其使用microRNA作为引物，并在DNA链延伸过程中掺入2′-脱氧腺苷-5 ′-O-（1-硫代三磷酸）作为dATP替代物的能力。研究了五种DNA聚合酶，包括嗜温DNA聚合酶I大Klenow片段、3′→5′ exo-Klenow DNA聚合酶、嗜热Bst DNA聚合酶大片段、Therminator™ DNA聚合酶和Taq DNA聚合酶。实验结果表明，除Taq DNA聚合酶外，其他聚合酶均能利用microRNA作为引物，并具有核苷酸类似物掺入和microRNA识别的共性和差异性。DNA聚合酶I的大Klenow片段显示没有可检测的聚合产物与硫代修饰的dATP作为底物。嗜热Bst DNA聚合酶对DNA模板上的microRNA识别具有最高的特异性。该研究为microRNA检测提供了一种新的方法，而无需逆转录为cDNA，该方法更快，更简单，更不容易出现偏差和错误。
MicroRNAs as endogenous regulators of gene expression have spurred a surge of interest for their quantification and expression analysis. High-sensitivity and specificity microRNA detection techniques such as Real-Time PCR and recently introduced bioluminescent microRNA detection require systematic study of DNA polymerases for use with microRNAs. In this study, a variety of DNA polymerases have been studied to assess their capabilities of using microRNA as a primer and incorporating 2′-deoxyadenosine-5′-O-(1-thiotriphosphate) as a dATP alternative during DNA strand extension. Five DNA polymerases were investigated including the mesophilic DNA polymerase I large Klenow fragment, 3′→5′ exo− Klenow DNA polymerase, thermophilic Bst DNA polymerase large fragment, Therminator™ DNA polymerase, and Taq DNA polymerase. The experimental results show that except for Taq DNA polymerase, the polymerases can utilize microRNA as a primer and have both common and divergent properties of the nucleotide analog incorporation and microRNA discrimination. DNA polymerase I large Klenow fragment showed no detectable polymerization product with the thio-modified dATP as a substrate. Thermophilic Bst DNA polymerase had the highest specificity for microRNA recognition on a DNA template. The study provides a novel method for microRNA detection without reverse transcription to cDNA, which is faster, simpler, and less prone to biases and errors.
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影响因子：
14.9
作者：
Chen C;Ridzon DA;Broomer AJ;Zhou Z;Lee DH;Nguyen JT;Barbisin M;Xu NL;Mahuvakar VR;Andersen MR;Lao KQ;Livak KJ;Guegler KJ
通讯作者：
Guegler KJ
影响因子：
56.9
作者：
Jopling, CL;Yi, MK;Sarnow, P
通讯作者：
Sarnow, P
影响因子：
2.9
作者：
Alexandre, I;Hamels, S;Remacle, J
通讯作者：
Remacle, J
影响因子：
2.9
作者：
Gharizadeh, B;Nordström, T;Nyrén, P
通讯作者：
Nyrén, P
影响因子：
3.2
作者：
Rajeevan, MS;Dimulescu, IM;Vernon, SD
通讯作者：
Vernon, SD