JaLCDOI 10.18926/AMO/32639
フルテキストURL fulltext.pdf
著者 Zhang, Bo| Seki, Shuji| Akiyama, Kosuke| Tsutsui, Ken| Li, Ting| Nagao, Kazutaka|
抄録 <p>DNA damage induced by cis-diamminedichloroplatinum (II) (cisplatin: cis-DDP), an anticancer drug, was studied in vitro by monitoring the drug-induced conformational change of pUC18 plasmid DNA, the sensitivity to some restriction enzymes of the damaged DNA and the sequence-dependent termination of DNA synthesis caused by cisplatin. Closed circular, superhelical pUC18 DNA was treated at 37 degrees C for 16 h with various concentrations of cisplatin. Cisplatin-dose-dependent conformational change due to unwinding of the treated DNA was detected by agarose gel electrophoresis. To analyze the base-specificity of the cisplatin damage, the measurement for sensitivity of cisplatin-treated DNA to various types of restriction enzyme and sequence gel analysis of the treated DNA were conducted. The results suggested that cisplatin attacked preferentially the sequence of GG &#62; AG &#62; GNG in the order. In the present assay condition, the cisplatin/DNA nucleotide ratios required for the DNA damage detection were roughly 0.025 for the conformational analysis, 0.001 or more for the restriction enzyme analysis, and less than 0.001 for the sequence gel analysis. By using the present method, it was demonstrated that the cisplatin-mediated DNA damage was inhibited by NaCl, KCl, CaCl2 or MgCl2 at their nearly physiological concentrations, and by reducing agents such as thiourea and 2-mercaptoethanol in the reaction mixture.</p>
キーワード DNA damage cisplatin gel electrophoresis sequence gel analysis
Amo Type Article
発行日 1992-12
出版物タイトル Acta Medica Okayama
46巻
6号
出版者 Okayama University Medical School
開始ページ 427
終了ページ 434
ISSN 0386-300X
NCID AA00508441
資料タイプ 学術雑誌論文
言語 English
論文のバージョン publisher
査読 有り
PubMed ID 1336637
Web of Sience KeyUT A1992KE49600004
JaLCDOI 10.18926/AMO/30857
フルテキストURL fulltext.pdf
著者 Ikeda, Shogo| Yamamoto, Mihoko| Nagao, Kazutaka| Zhang, Bo| Watanabe, Sekiko| Oda, Takuzo|
抄録 <p>Non-radioactive hybridization probes were prepared using the M13 phage vector and the universal sequencing primer. The probe sequence to be used was first cloned into the M13 vector, and the minus strand of the template DNA was then synthesized with the Klenow fragment of E. coli DNA polymerase I in the presence of the biotinylated nucleotide, biotin-11-dUTP, as a label. Resultant DNA was heavily biotinylated, and made up of the entire minus strand of the template DNA. The long tag sequence derived from the M13 vector may increase the sensitivity of the detection. The biotinylated hybrids were visualized with the streptavidin-alkaline phosphatase conjugate and chromogenic substrates. As shown by Southern hybridization, the probe prepared in this way could be used to detect less than 1 pg of target sequence and a single copy gene sequence in human genomic DNA within several hours of signal development.</p>
キーワード non-rradioctive probe biotin nucleotide M13 phage DNA universal sequencing primer Southern hybridization
Amo Type Article
発行日 1989-08
出版物タイトル Acta Medica Okayama
43巻
4号
出版者 Okayama University Medical School
開始ページ 197
終了ページ 202
ISSN 0386-300X
NCID AA00508441
資料タイプ 学術雑誌論文
言語 English
論文のバージョン publisher
査読 有り
PubMed ID 2678902
Web of Sience KeyUT A1989AP79100001