基于HIV-1逆转录酶的细胞脱氧核苷酸三磷酸浓度测定法。
HIV-1 Reverse Transcriptase-Based Assay to Determine Cellular dNTP Concentrations.
作者信息
Hollenbaugh Joseph A, Kim Baek
机构信息
Center for Drug Discovery, Emory Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, 1760 Haygood Drive, Health Sciences Research Building, Atlanta, GA, 30322, USA.
College of Pharmacy, Kyung-Hee University, Seoul, South Korea.
出版信息
Methods Mol Biol. 2016;1354:61-70. doi: 10.1007/978-1-4939-3046-3_5.
Abstract
Deoxynucleoside triphosphates (dNTPs) are the building blocks of DNA and their biosynthesis is tightly regulated in the cell. HPLC-MS and enzyme-based methods are currently employed to determine dNTP concentrations from cellular extracts. Here, we describe a highly efficient, HIV-1 reverse transcriptase (RT)-based assay to quantitate dNTP concentrations. The assay is based on the ability of HIV-1 RT to function at very low dNTP concentrations, thus providing for the high sensitivity of detection.
摘要
脱氧核苷三磷酸（dNTPs）是DNA的组成成分，其生物合成在细胞中受到严格调控。目前采用高效液相色谱-质谱联用（HPLC-MS）和基于酶的方法来测定细胞提取物中的dNTP浓度。在此，我们描述了一种基于高效HIV-1逆转录酶（RT）的检测方法来定量dNTP浓度。该检测方法基于HIV-1 RT在极低dNTP浓度下仍能发挥作用的能力，从而实现了高灵敏度检测。
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