title: PCR synthesis of double stranded DNA labeled with 5-bromouridine. A step towards finding a bromonucleoside for clinical trials.
abstract: Incorporation of 5-bromouridine (5BrdU) into DNA makes it sensitive to UV and ionizing radiation, which opens up a prospective route for the clinical usage of 5-bromouridine and other halonucleosides. In the present work the polymerase chain reaction (PCR) protocol, which enables a long DNA fragment (resembling DNA synthesized in the cell in the presence of halonucleosides) to be completely substituted with 5BrdU, was optimized. Using HPLC coupled to enzymatic digestion, it was demonstrated that the actual amounts of native nucleosides and 5BrdU correspond very well to those calculated from the sequence of PCR products. The synthesized DNA is photosensitive to photons of 300nm. HPLC analysis demonstrated that the photolysis of labeled PCR products leads to a significant decrease in the 5BrdU signal and the simultaneous occurrence of a uridine peak. Agarose and polyacrylamide gel electrophoresis suggest that single strand breaks and cross-links are formed as a result of UV irradiation. The PCR protocol described in the current paper may be employed for labeling DNA not only with BrdU but also with other halonucleosides.
authors.affiliation: Department of Chemistry, University of Gdańsk, Sobieskiego 18, 80-952 Gdańsk, Poland.
authors.forename: Barbara
authors.initials: B
authors.lastname: Michalska
authors.forename: Ireneusz
authors.initials: I
authors.lastname: Sobolewski
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Polska
authors.forename: Justyna
authors.initials: J
authors.lastname: Zielonka
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Zylicz-Stachula
authors.forename: Piotr
authors.initials: P
authors.lastname: Skowron
authors.forename: Janusz
authors.initials: J
authors.lastname: Rak
mesh_terms: D001976:Bromouracil / Q000031:analogs & derivatives; D002986:Clinical Trials as Topic; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000032:analysis / Q000378:metabolism; D004339:Drug Compounding / Q000379:methods*; D004353:Drug Evaluation, Preclinical; D054879:Halogenation; D006801:Humans; D009705:Nucleosides / Q000737:chemistry / Q000378:metabolism; D009841:Oligonucleotides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D017319:Photosensitizing Agents / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D014529:Uridine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D014544:Uridine Triphosphate / Q000737:chemistry / Q000378:metabolism
chemical_list: D009705:Nucleosides; D009841:Oligonucleotides; D017319:Photosensitizing Agents; D001976:Bromouracil; D004247:DNA; D004259:DNA-Directed DNA Polymerase; C006824:5-bromouridine; D014544:Uridine Triphosphate; D014529:Uridine