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 Scholarshi
