HIV-1 Reverse Transcriptase-Based Assay to Determine Cellular dNTP Concentrations

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.

Keywords: Deoxynucleoside triphosphates; HIV-1; Reverse transcriptase; dNTP concentration.

Figure 1. HIV RT-based dNTP urea-PAGE analysis

Sample reactions were resolved on a 14% urea-PAGE. Imaging Screen K (BioRad) was exposed to the dried gel. The image data were captured using PharosFX Plus Molecular Imager (BioRad). Data analysis was accomplished by using QuantityOne software (BioRad). The unextended primer (Primer) and the extended primer (Primer +1) are shown. Lanes 1 and 2 are the negative and positive controls, respectively. Lane 3 was left empty. Lanes 4–8 were the samples to be quantitated. A quantity box was set to capture the raw data. U1, negative control (Primer +1 area) was used to subtract background. Boxes U2–U6 were for the unextended Primer values, whereas boxes U7–U11 captured data for extended Primer +1 values.