Prokaryotic and eukaryotic organisms require coordinated regulation of intracellular deoxyribonucleoside triphosphate (dNTP) pools to maintain the fidelity of DNA synthesis during DNA replication and repair. Dysregulation of intracellular dNTP pools occurs in many diseases and is exploited by some pharmacological inhibitors. Therefore, quantification of cellular dNTP levels is an essential element for understanding the biological changes that result in altered dNTP biosynthesis and the mechanisms of action of pharmacological agents.
Current methods for measuring dNTPs use hazardous reagents in cellular extraction protocols and require the use of radioactivity in labor-intensive DNA polymerase–based assays and/or chromatography-based procedures. As an alternative to these techniques, the authors describe a rapid and sensitive fluorescence-based method that uses synthetic templates (IDT oligonucleotides), a PrimeTime® Primer, and ZEN™ Double-Quenched Probes for quantifying cellular dNTPs. This new method, which is compatible with methanol-based dNTP isolation and performed on a standard real-time PCR thermal cycler, eliminates the need for hazardous reagents and specialized chromatography and mass spectrometry instruments.