Adapting capillary gel electrophoresis as a sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes.
Adapting capillary gel electrophoresis as a sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes.
复制标题
DOI：
10.1093/nar/gkv899
复制
发表时间：
2016-01-29
影响因子：
14.9
通讯作者：
Gardner AF
中科院分区：
文献类型：
--
作者：
Greenough L;Schermerhorn KM;Mazzola L;Bybee J;Rivizzigno D;Cantin E;Slatko BE;Gardner AF
核酸酶的详细生化表征是理解核酸代谢、基因组复制和修复的基础。我们报告了一种快速、高通量荧光毛细管凝胶电泳方法的开发，作为传统聚丙烯酰胺凝胶电泳的替代方法来表征核酸代谢酶。这里描述的测定设计原理可以应用于几乎任何作用于荧光标记寡核苷酸底物的酶系统。在此，我们描述了使用这种核心毛细管凝胶电泳方法来加速核酸酶研究的几种测定。首先，设计检测方法来检查 DNA 聚合酶活性，包括核苷酸掺入动力学、链置换合成和 3'-5' 核酸外切酶活性。接下来，监测DNA连接酶、活瓣核酸内切酶和RNase H2的DNA修复活性。此外，还进行了在单个反应中使用四种不同荧光标记底物的多色测定来表征 GAN 核酸酶特异性。最后，描述了用于监测冈崎片段成熟过程中偶联酶反应的双色荧光测定。这些测定作为模板，指导以高通量方式进行酶表征或核苷和非核苷抑制剂筛选的进一步技术开发。
Detailed biochemical characterization of nucleic acid enzymes is fundamental to understanding nucleic acid metabolism, genome replication and repair. We report the development of a rapid, high-throughput fluorescence capillary gel electrophoresis method as an alternative to traditional polyacrylamide gel electrophoresis to characterize nucleic acid metabolic enzymes. The principles of assay design described here can be applied to nearly any enzyme system that acts on a fluorescently labeled oligonucleotide substrate. Herein, we describe several assays using this core capillary gel electrophoresis methodology to accelerate study of nucleic acid enzymes. First, assays were designed to examine DNA polymerase activities including nucleotide incorporation kinetics, strand displacement synthesis and 3′-5′ exonuclease activity. Next, DNA repair activities of DNA ligase, flap endonuclease and RNase H2 were monitored. In addition, a multicolor assay that uses four different fluorescently labeled substrates in a single reaction was implemented to characterize GAN nuclease specificity. Finally, a dual-color fluorescence assay to monitor coupled enzyme reactions during Okazaki fragment maturation is described. These assays serve as a template to guide further technical development for enzyme characterization or nucleoside and non-nucleoside inhibitor screening in a high-throughput manner.