| Mismatched Primer Extension Assays
|
|
Author:
Date:
2015-06-20
[Abstract] Steady state kinetic assays have been a reliable way to estimate fidelity of several polymerases (Menendez-Arias, 2009; Rezende and Prasad, 2004; Svarovskaia et al., 2003). The ability to analyze the extension of primers with specific mismatches at the 3ʹ end is a major strength of the mismatched primer extension assays. Recently, we used the mismatched primer extension assays to show that the fidelity of HIV RT increases dramatically when concentration of Mg2+ is reduced to a physiologically relevant concentration (~0.25 mM) (Achuthan et al., 2014). Here, we describe in detail how to perform the mismatched primer extension assay to measure the standard extension efficiency using human immunodeficiency virus reverse transcriptase (HIV RT) at 2 mM Mg2+ ...
[摘要] 稳态动力学测定法是估计几种聚合酶的保真度的可靠方法（Menendez-Arias，2009; Rezende和Prasad，2004; Svarovskaia等人，2003）。分析具有在3'末端的特异性错配的引物的延伸的能力是错配引物延伸测定的主要强度。最近，我们使用错配的引物延伸测定法显示当Mg 2+浓度降低到生理相关浓度（〜0.25mM）时，HIV RT的保真度显着增加（Achuthan等al 。，2014）。在这里，我们详细地描述如何进行错配引物延伸测定以使用人类免疫缺陷病毒逆转录酶（HIV RT）在2mM Mg 2+ 2 + 测量标准延伸效率。然后可以使用标准延伸效率估计聚合酶的相对保真度。这里描述的测定基于在Mendelman等人（1990）中公开的方法。
|
|
|
| Primer Extension Reactions for the PCR- based α- complementation Assay
|
|
Author:
Date:
2015-06-20
[Abstract] The PCR- based- α- complementation assay is an effective technique to measure the fidelity of polymerases, especially RNA-dependent RNA polymerases (RDRP) and Reverse Transcriptases (RT). It has been successfully employed to determine the fidelity of the poliovirus polymerase 3D-pol (DeStefano, 2010) as well as the human immunodeficiency virus Reverse Transcriptase (HIV RT) (Achuthan et al., 2014). A major advantage of the assay is that since the PCR step is involved, even the low yield of products obtained after two rounds of low yield of RNA synthesis (for RDRP) or reverse transcription (for RT) can be measured using the assay. The assay also mimics the reverse transcription process, since both RNA- and DNA- directed RT synthesis steps are performed. We recently used this assay ...
[摘要] 基于PCR的α-互补分析是测量聚合酶，特别是RNA依赖性RNA聚合酶（RDRP）和逆转录酶（RT）的保真度的有效技术。它已经成功地用于确定脊髓灰质炎病毒聚合酶3D-pol（DeStefano，2010）以及人类免疫缺陷病毒逆转录酶（HIV RT）的保真度（Achuthan等人，2014）。该测定法的主要优点是，由于涉及PCR步骤，甚至可以使用该测定法测量在两轮低合成RNA合成（对于RDRP）或逆转录（对于RT）后获得的产物的低产率。该测定也模拟逆转录过程，因为进行RNA和DNA指导的RT合成步骤。我们最近使用该测定法显示在生理相关镁浓度下的HIV RT具有与其它逆转录酶相同范围内的准确度（Achuthan等人，2014）。在这里，我们详细描述如何使用引物延伸反应准备插入。然后在基于PCR的α-互补分析中进一步处理制备的插入片段。
|
|
|