ArticleNucleic acids research2011
A novel fluorescence-based assay for the rapid detection and quantification of cellular deoxyribonucleoside triphosphates.
Article in Nucleic acids research, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 51 papers.
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Who cites it
51 citing papers in PubMed, 91 citations in OpenAlex.
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- Altered S-AdenosylMethionine availability impacts dNTP pools in Saccharomyces cerevisiae.Yeast (Chichester, England) · 2024Article
- Using Selective Enzymes to Measure Noncanonical DNA Building Blocks: dUTP, 5-Methyl-dCTP, and 5-Hydroxymethyl-dCTP.Biomolecules · 2023Article
- Quantitative Analysis of Nucleoside Triphosphate Pools in Mouse Muscle Using Hydrophilic Interaction Liquid Chromatography Coupled with Tandem Mass Spectrometry Detection.Methods in molecular biology (Clifton, N.J.) · 2023Article
- dTMP imbalance through thymidylate 5'-phosphohydrolase activity induces apoptosis in triple-negative breast cancers.Scientific reports · 2022Article
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- Isocratic HPLC analysis for the simultaneous determination of dNTPs, rNTPs and ADP in biological samples.Nucleic acids research · 2022Article
- Enzymatic deamination of the epigenetic nucleoside N6-methyladenosine regulates gene expression.Nucleic acids research · 2021Article
- A Magnetic Modulation Biosensing-Based Molecular Assay for Rapid and Highly Sensitive Clinical Diagnosis of Coronavirus Disease 2019 (COVID-19).The Journal of molecular diagnostics : JMD · 2021Article
- Equine lentivirus counteracts SAMHD1 restriction by Rev-mediated degradation of SAMHD1 via the BECN1-dependent lysosomal pathway.Autophagy · 2021Article
- Targeting nucleotide metabolism enhances the efficacy of anthracyclines and anti-metabolites in triple-negative breast cancer.NPJ breast cancer · 2021Article
- dUTPase inhibition confers susceptibility to a thymidylate synthase inhibitor in DNA-repair-defective human cancer cells.Cancer science · 2021Article
- A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye and inhibitor-resistant high-fidelity DNA polymerase.Nucleic acids research · 2020Article
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Authors and funding
6 authors at 2 institutions in 1 country.
Funding
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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