What is it about?

During our studies of pyrimidine nucleotide metabolism in mice, we encountered the problem that there was no suitable method to measure dNTPs from tissue samples. We first tested a fluoroenzymatic method originally developed for cultured cells, but this lacked the robustness and sensitivity required to quantify the low concentration of dNTPs in complex tissue extracts. Therefore, we developed a novel highly sensitive assay based on long synthetic DNA oligonucleotides, EvaGreen dye and an inhibitor-resistant high-fidelity DNA polymerase. We also optimized the extraction to minimize impurities that, critically, interfere with the detection. Moreover, one of the technical challenges was the disturbance potentially caused by ribonucleotides, the building blocks of RNA that may be thousands of times more abundant than dNTPs in tissues. Developing the technique, PhD student Janne Purhonen realized that the addition of another enzyme (RNAse HII) that cuts the DNA strand at an erroneously added ribonucleotide allows differentiation of the correct and false reaction products based on their melting temperature. This way, the signal due to erroneously incorporated ribonucleotides, if any, could be removed. Our assay is simple, non-radioactive, inexpensive, and most importantly has the sensitivity and robustness to measure minute dNTP concentrations from tissues comprising mostly non-proliferating cells.

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Why is it important?

Only a handful of studies have addressed the tissue concentration of dNTPs in health and disease, presumably because of lack of sensitive-enough methodology applicable in any laboratory with basic molecular biology equipment. Therefore, our new method fills a methodological gap and will facilitate the understanding of regulation and fluctuations in dNTP pools in normal biology and in disease settings. We hope the assay will turn out useful to researchers of mitochondrial diseases as well as elsewhere in biological and medical research.

Perspectives

This project was mainly an innovation by our PhD student Janne Purhonen. Another PhD student, Rishi Banerjee, was responsible for the cell culture experiments. Dr Allison MacDonald from Wilfrid Lauriel University, Canada, kindly provided the Pacific oyster alternative oxidase (AOX) cDNA, which her group had previously shown in yeast to bypass respiratory chain blockade. In this paper, we present the first data on heterologous expression of Pacific oyster AOX in mammalian cells.

Dr Jukka Kallijärvi
Folkhälsan Research Center

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This page is a summary of: A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye and inhibitor-resistant high-fidelity DNA polymerase, Nucleic Acids Research, June 2020, Oxford University Press (OUP),
DOI: 10.1093/nar/gkaa516.
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