title: A novel fluorescence-based assay for the rapid detection and quantification of cellular deoxyribonucleoside triphosphates.
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.
authors.affiliation: Department of Pathology, University of Southern California, Los Angeles, CA 90089, USA.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Wilson
authors.forename: Melissa J
authors.initials: MJ
authors.lastname: Labonte
authors.forename: Jared
authors.initials: J
authors.lastname: Russell
authors.forename: Stan
authors.initials: S
authors.lastname: Louie
authors.forename: Andrew A
authors.initials: AA
authors.lastname: Ghobrial
authors.forename: Robert D
authors.initials: RD
authors.lastname: Ladner
mesh_terms: D004259:DNA-Directed DNA Polymerase; D003854:Deoxyribonucleotides / Q000032:analysis*; D003856:Deoxyuracil Nucleotides / Q000032:analysis; D031541:Fluorescence Resonance Energy Transfer; D045325:HCT116 Cells; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012265:Ribonucleotides / Q000737:chemistry; D013698:Templates, Genetic
chemical_list: D003854:Deoxyribonucleotides; D003856:Deoxyuracil Nucleotides; D015345:Oligonucleotide Probes; D012265:Ribonucleotides; C027078:deoxyuridine triphosphate; D004259:DNA-Directed DNA Polymerase