A novel fluorescence-based assay for the rapid detection and quantification of cellular deoxyribonucleoside triphosphates - PubMed (original) (raw)

A novel fluorescence-based assay for the rapid detection and quantification of cellular deoxyribonucleoside triphosphates

Peter M Wilson et al. Nucleic Acids Res. 2011.

Abstract

Current methods for measuring deoxyribonucleoside triphosphates (dNTPs) employ reagent and labor-intensive assays utilizing radioisotopes in DNA polymerase-based assays and/or chromatography-based approaches. We have developed a rapid and sensitive 96-well fluorescence-based assay to quantify cellular dNTPs utilizing a standard real-time PCR thermocycler. This assay relies on the principle that incorporation of a limiting dNTP is required for primer-extension and Taq polymerase-mediated 5-3' exonuclease hydrolysis of a dual-quenched fluorophore-labeled probe resulting in fluorescence. The concentration of limiting dNTP is directly proportional to the fluorescence generated. The assay demonstrated excellent linearity (R(2) > 0.99) and can be modified to detect between ∼0.5 and 100 pmol of dNTP. The limits of detection (LOD) and quantification (LOQ) for all dNTPs were defined as <0.77 and <1.3 pmol, respectively. The intra-assay and inter-assay variation coefficients were determined to be <4.6% and <10%, respectively with an accuracy of 100 ± 15% for all dNTPs. The assay quantified intracellular dNTPs with similar results obtained from a validated LC-MS/MS approach and successfully measured quantitative differences in dNTP pools in human cancer cells treated with inhibitors of thymidylate metabolism. This assay has important application in research that investigates the influence of pathological conditions or pharmacological agents on dNTP biosynthesis and regulation.

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Figures

Figure 1.

Figure 1.

Simplified schematic illustrating the principle mechanism involved in the fluorescence-based assay for measuring dNTP concentrations. Detection of dTTP using template DT6 is given as the example. The template is depicted in blue, the FAM-dTTP probe in green and primer NDP1 in red. Briefly, as the temperature declines from the 95°C hot-start, the probe anneals to the template first (65–70°C), followed by the primer at 60°C to form the TPP complex at which point Taq polymerase begins extension of the nascent strand. In the presence of a sufficient concentration of limiting dNTP (six dTTP molecules in the example of dTTP-DT6), successful primer extension occurs through the mid-template dNTP detection region and Taq polymerase cleaves the terminal nucleotide labeled with the 6-FAM fluorophore via its 5′-3′ exonuclease activity releasing it from the dual-quenched (ZEN and IBFQ) probe resulting in disruption of FRET and generation of a fluorescence signal in response to excitation-induced photon energy (h_v_). When the dNTP being measured (dTTP) is not present or becomes exhausted, Taq polymerase stalls, extension is inhibited/terminated, fluorescence remains quenched via FRET and the probe remains dark. In any given reaction, the level of fluorescence generated is directly proportional to the concentration of the limiting dNTP. The dAMP molecules enlarged in the template strand represent the nucleotides opposite which the limiting dTTP nucleotides (also enlarged and in bold) will base pair. Only the nucleotide sequence found in the mid-template dNTP detection region is given for simplicity. The complete sequences of all templates (including primer- and probe-binding regions), primer NDP1 and detection probes are given in Table 1. Template, primers and probes were prepared as described in ‘Materials and Methods’ section.

Figure 2.

Figure 2.

Validation of dTTP templates with varying detection sensitivities and linear range. Three specific oligonucleotide templates were initially generated and tested for their ability to detect dTTP and tested by calibration curve as described in ‘Materials and Methods’ section. (A) dTTP-DT6 requires the incorporation of six dTTPs for fluorescence generation and yielded a linear range of 0–100 pmol. (B) dTTP-DT2 requires the incorporation of two dTTPs and yielded a linear range of 0–25 pmol. (C) Finally, dTTP-DT1 requires only a single dTTP for incorporation per TPP complex to yield fluorescence and had a linear range of 0.6–10 pmol. Calibration curves for all three templates demonstrated _R_2 of >0.993. In all cases, fluorescence values for blank reactions (limiting dNTP omitted) were subtracted to give NFU.

Figure 3.

Figure 3.

Validation of dATP, dCTP and dGTP detection templates. Calibration curves were generated for dNTPs using dNTP-specific templates (DT1 and DT2) and probes (Table 1) and were performed as described in ‘Materials and Methods’ section. In all cases, fluorescence values for blank reactions (limiting dNTP omitted) were subtracted to give NFU. All curves demonstrated _R_2 > 0.99. (A) dATP, (B) dCTP, (C) dGTP. Left, DT1; Right, DT2.

Figure 4.

Figure 4.

Time-course and calibration curve analysis of the polymerase reaction. Time course showing fluorescence generated by the dNTP-dependent Taq DNA polymerase-mediated hydrolysis of a dual-quenched fluorescent-labeled probe. Left. Known pmole quantities of dNTP were detected using DT1 and fluorescence was analyzed at 5-min intervals on board an Applied Biosystems 7500 Real-Time PCR System. Right. Calibration curves were generated and plotted from the NFU obtained at the specified time intervals and analyzed by linear regression. All calibration curves demonstrated _R_2 of >0.99. (A) dGTP. (B) dTTP. (C) dATP. (D) dCTP. Additional details of the assay are described in the ‘Materials and Methods’ section.

Figure 5.

Figure 5.

Effect of a 100- and 1000-fold molar rNTP excess on the recovery of dNTPs in the fluorescence-based assay with Taq polymerase. (A) The recovery of 5 pmol of dGTP was determined in the presence of both a 100- and 1000-fold molar excess of GTP at assay completion (20 min). The same analysis was applied to the recovery of (B) dATP, (C) dCTP and (D) dTTP in the presence of their corresponding rNTP. Bars represent the mean + SD of three individual analyses. The assay was performed as described in ‘Materials and Methods’ section. In all cases, fluorescence values for blank reactions (limiting dNTP omitted) were subtracted to give NFUs.

Figure 6.

Figure 6.

Detection of dUTP. The ability of the assay to detect dUTP and distinguish dTTP from dUTP in the presence and absence of the dUTP-hydrolyzing enzyme dUTPase was analyzed. (A) The effects of including recombinant human dUTPase (DUT) and a 5 min pre-incubation at 37°C were first analyzed. Inclusion of 5 ng of dUTPase had no significant impact on the assay performance and detection of dTTP (_R_2 > 0.99). (B) dTTP was replaced with dUTP and the reaction performed in the absence of dUTPase and in the presence of 2.5 and 5 ng of recombinant human dUTPase. In the absence of dUTPase, dUTP detection was robust and yielded an excellent calibration curve (_R_2 > 0.99). Five nanograms of dUTPase was sufficient to eliminate dUTP as the source of fluorescence in the assay, whereas 2.5 ng resulted in partial hydrolysis and intermediate fluorescence. The assay was performed as described in ‘Materials and Methods’ section. In all cases, fluorescence values for blank reactions (limiting dNTP omitted) were subtracted to give NFUs.

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