An improved and widely accessible dNTP quantitation tool

Szabó, Judit Eszter and Mébold, Bence and Surányi, Éva Viola and Vértessy, Beáta and Cserepes, Mihály and Szakács, Gergely and Dobay, Orsolya and Szabó, Dóra and Tóth, Judit (2018) An improved and widely accessible dNTP quantitation tool. In: FEBS3+ From molecules to living systems, 2-5 September 2018, Siófok, Hungary.

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Abstract

Cells maintain a fine-tuned concentration balance in the pool of deoxyribonucleoside 5’- triphosphates (dNTPs). The perturbation of this balance results in increased mutation frequencies suggested to promote cancer development and drug resistance. To study dNTP imbalances and their consequences, an accurate and relatively high-throughput method is necessary. The dNTP quantitation method of our choice is a fluorescence-based, TaqMan-like polymerase assay published by Wilson et al, NAR 2011. This assay has the advantages of being accessible in a standard molecular biology laboratory and having the potential to be automated in contrast to mass spectrometry or radioactive measurements. Although this method works well in diluted samples with high dNTP levels, we observed that the sample matrix largely decreases assay performance. Upon thorough kinetic analysis of the fluorescent dNTP incorporation curves, we found that the Taq polymerase exhibits a dNTP independent, signal generating exonuclease activity and that the polymerization and exonuclease activity are partially inhibited by the sample matrix. Based on our kinetic investigations we suggest several assay modifications and a novel, kineticsbased and automated analysis method. Using these modifications, we measured dNTP pools in widely different organisms including Mycobacterium smegmatis, Staphylococcus aureus and human cancer cells. We found that our improved method is capable of i) determining dNTP concentrations in samples previously proved to be unmeasurable by eliminating the interfering matrix effect, and ii) improving the quantitation limits of the assay. Fundings: NKFIH-PD 124330, NKFIH-K 115993, János Bolyai Research Scholarship

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