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ID 30857
JaLCDOI
フルテキストURL
著者
Ikeda, Shogo Okayama University
Yamamoto, Mihoko Okayama University
Nagao, Kazutaka Okayama University
Zhang, Bo Okayama University
Watanabe, Sekiko Okayama University
Oda, Takuzo Okayama University
抄録

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.

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
Web of Sience KeyUT