Abstract
Deoxyribonucleoside triphosphates (dNTPs) are vital for the biosynthesis and repair of DNA. Their cellular concentration peaks during the S phase of the cell cycle. In non-proliferating cells, dNTP concentrations are low, making their reliable quantification from tissue samples of heterogeneous cellular composition challenging. Partly because of this, the current knowledge related to the regulation of and disturbances in cellular dNTP concentrations derive mostly from cell culture experiments with little corroboration at the tissue or organismal level. Here, we fill the methodological gap by presenting a simple non-radioactive microplate assay for the quantification of dNTPs with a minimum requirement of 4–12 mg of biopsy material. In contrast to published assays, this assay is based on long synthetic single-stranded DNA templates (50–200 nucleotides), an inhibitor-resistant high-fidelity DNA polymerase, and the double-stranded-DNA-binding EvaGreen dye. The assay quantified reliably less than 50 fmol of each of the four dNTPs and discriminated well against ribonucleotides. Additionally, thermostable RNAse HII-mediated nicking of the reaction products and a subsequent shift in their melting temperature allowed near-complete elimination of the interfering ribonucleotide signal, if present. Importantly, the assay allowed measurement of minute dNTP concentrations in mouse liver, heart and skeletal muscle.
Highlights
Deoxyribonucleoside triphosphates are building blocks of DNA and, their cellular concentration is highest during active DNA synthesis, such as in proliferating cells [1]
Balanced Deoxyribonucleoside triphosphates (dNTPs) pools are vital for efficient error-free DNA synthesis [1]
The current knowledge related to regulation and fluctuations in dNTP pools derive mainly from cell culture experiments
Summary
Deoxyribonucleoside triphosphates (dNTPs) are building blocks of DNA and, their cellular concentration is highest during active DNA synthesis, such as in proliferating cells [1]. Traditional enzymatic methods to measure dNTPs have relied on the incorporation of radioactive dNTPs to a complementary strand of a DNA template by a DNA polymerase [4,5] These methods have mainly been used to measure dNTPs from actively proliferating cultured cells with high dNTP concentrations and a limited complexity of interfering biological matrix. A simple probe hydrolysis-based enzymatic fluorometric assay has been developed to measure dNTPs from cultured cells [7]. This assay utilizes synthetic DNA templates with a 3 primer-binding region, mid-template dNTP-detection region, 5 probe-binding sequence, and a Taq DNA polymerase with 3 to 5 exonuclease activity to hydrolyse the probe - an adaptation of typical TaqMan qPCR chemistry. Due to the simplicity of this fluorometric assay, we set out to test it as a means to quantify dNTPs in mouse
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