A user-friendly, high-throughput tool for the precise fluorescent quantification of deoxyribonucleoside triphosphates from biological samples.

Bence Sándor Mébold,Judit Eszter Szabó,Judit Tóth,Mihály Cserepes,Tamás Trombitás,Éva Viola Surányi

doi:10.1093/nar/gkaa116

Abstract

Cells maintain a fine-tuned, dynamic concentration balance in the pool of deoxyribonucleoside 5′-triphosphates (dNTPs). This balance is essential for physiological processes including cell cycle control or antiviral defense. Its perturbation results in increased mutation frequencies, replication arrest and may promote cancer development. An easily accessible and relatively high-throughput method would greatly accelerate the exploration of the diversified consequences of dNTP imbalances. The dNTP incorporation based, fluorescent TaqMan-like assay published by Wilson et al. has the aforementioned advantages over mass spectrometry, radioactive or chromatography based dNTP quantification methods. Nevertheless, the assay failed to produce reliable data in several biological samples. Therefore, we applied enzyme kinetics analysis on the fluorescent dNTP incorporation curves and found that the Taq polymerase exhibits a dNTP independent exonuclease activity that decouples signal generation from dNTP incorporation. Furthermore, we found that both polymerization and exonuclease activities are unpredictably inhibited by the sample matrix. To resolve these issues, we established a kinetics based data analysis method which identifies the signal generated by dNTP incorporation. We automated the analysis process in the nucleoTIDY software which enables even the inexperienced user to calculate the final and accurate dNTP amounts in a 96-well-plate setup within minutes.

Highlights

Despite the longstanding need and desire for the precise quantification of cellular levels of deoxyribonucleoside triphosphates, none of the existing methods could become routine molecular biology procedure
As this is far more the most attainable method to measure deoxyribonucleoside triphosphates (dNTPs) concentrations in a standard molecular biology laboratory, we set out to explore the background of this phenomenon and modify the assay so that it can be used in any biological samples
The calculation method they used assumed that the fluorescence intensity at a given time point should be used to calculate the limiting dNTP concentration in the sample using a linear calibration curve measured in water

Summary

Introduction

Despite the longstanding need and desire for the precise quantification of cellular levels of deoxyribonucleoside triphosphates (dNTP), none of the existing methods could become routine molecular biology procedure. To understand the role of dNTP concentration changes in these processes, the most valuable information could be obtained by measuring intracellular dNTP levels directly in the cell. As no method is available to do so, we would need to reliably evaluate the dNTP concentration of cellular lysates, at least. Each method has its advantages and disadvantages but in general, the biggest challenge of dNTP quantitation is that other nucleotides, primarily ribonucleoside triphosphates (rNTPs) are present in much higher concentration in the cell lysate than dNTPs and the separation of the various nucleotides from each other and from the sample matrix is not straightforward

Methods

Results

Conclusion