A one-step method for quantitative determination of uracil in DNA by real-time PCR
Keywords in Hungarian
DNS,PCR, Uracil
Keywords in English
DNA, PCR, Uracil
Discipline
General biochemistry and metabolism (Council of Medical and Biological Sciences)
100 %
Ortelius classification: Enzimology
Panel
Publications Panel
Department or equivalent
Institute of Enzymology, BRC, Hungarian Academy of Sciences
Starting date
2011-02-01
Closing date
2011-12-31
Funding (in million HUF)
0.300
FTE (full time equivalent)
0.00
state
closed project
Summary in Hungarian
Az uracil előfordulását a DNS-ben eredményezheti citozin dezamináció, illetve timint helyettesítő beépülés. A DNS-beli uracil kvantitatív mérésével olyan folyamatokat követhetünk nyomon, mint a DNS károsodása felborult timidilát metabolizmusú sejtekben, vagy az immunoglobulin diverzifikáció a megfelelő lókuszokban. Az archea Pyrococcus furiosus DNS polimeráza nagy affinitással köt olyan dezaminált bázisokhoz, mint az uracil, így megakad az uracilt tartalmazó templátok másolásánál. Ebben a tanulmányban bemutatunk egy közvetlen módszert, mely alkalmas a DNS uraciltartalmának kvantitatív meghatározására behatárolható genomi szegmensekre nézve. Ehhez a vad típusú Pyrococcus furiosus DNS polimerázát és annak pont mutáns, uracil felismerő tulajdonsággal nem rendelkező változatát párhuzamosan használjuk szintetikus vagy genomi DNS mintákon, hogy azok uraciltartalmát meghatározzuk egy lépéses real-time PCR analízissel. A real-time PCR eredmények kiértékelése hasonlóan végezhető, mint a különböző mintákból való a templát mennyiség összehasonlítása. Izotópos méréseink alátámasztották az uracilt tartalmazó mesterséges templátokból mért adatokat. A módszert teszteltük fiziológiás eredetű DNS mintákon, melyet felborult timidilát metabolizmussal rendelkező E. coli és egér sejtvonalból izoláltunk. Az itt bemutatott módszer lehetővé teszi az uraciltartalom egyszerű módon történő kimutatását olyan genomi szegmensekben, melyeket a primerekkel jelöltünk ki. Különböző primerpárokkal lehetővé válik az uracil eloszlás heterogenitása is a genomon belül.
Summary
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 Pyrococcus 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 E. 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.
Final report
Results in Hungarian
A jelen pályázat publikációs célú, a közlésre került cikk kivonata angolul:
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.
Results in English
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.
