A one-step method for quantitative determination of uracil in DNA by real-time PCR.
Uracil may occur in DNA due to either cytosine deamination or thymine replacing incorporation. Its quantitative characterization is important in assessing DNA damages in cells with perturbed thymidylate metabolism or within different DNA segments involved in immunoglobulin gene diversification. The archaeal DNA polymerase from Pyrococcus furiosus binds strongly to the deaminated base uracil and stalls on uracil-containing templates. Here, we present a straightforward method for quantitative assessment of uracil in DNA within specific genomic segments. We use wild-type P. furiosus polymerase in parallel with its point mutant version which lacks the uracil-binding specificity on synthetic and genomic DNA samples to quantify the uracil content in a single-step real-time PCR assay. Quantification of the PCR results is based on an approach analogous to template copy number determination in comparing different samples. Data obtained on synthetic uracil-containing templates are verified by direct isotopic measurements. The method is also tested on physiological DNA samples from Escherichia coli and mouse cell lines with perturbed thymidylate biosynthesis. The present PCR-based method is easy to use and measures the uracil content within a genomic segment defined by the primers. Using distinct sets of primers, the method allows the analysis of heterogeneity of uracil distribution within the genome.
Horváth
András
A
Laboratory of Genome Metabolism and Repair, Department of Applied Biotechnology, Institute of Enzymology, Hungarian Academy of Sciences, Budapest University of Technology and Economics, Budapest, Hungary.
Vértessy
Beáta G
BG
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000737:chemistry*; D004269:DNA, Bacterial / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D003857:Deoxyuridine / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D016680:Genome, Bacterial; D051379:Mice; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D020027:Pyrococcus furiosus / Q000201:enzymology; D013942:Thymine Nucleotides / Q000096:biosynthesis; D014498:Uracil / Q000032:analysis*
D004269:DNA, Bacterial; D013942:Thymine Nucleotides; D014498:Uracil; D004247:DNA; D004259:DNA-Directed DNA Polymerase; C024157:thymidine 5'-triphosphate; D003857:Deoxyuridine