JaLCDOI 10.18926/AMO/30857
FullText URL fulltext.pdf
Author Ikeda, Shogo| Yamamoto, Mihoko| Nagao, Kazutaka| Zhang, Bo| Watanabe, Sekiko| Oda, Takuzo|
Abstract <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>
Keywords non-rradioctive probe biotin nucleotide M13 phage DNA universal sequencing primer Southern hybridization
Amo Type Article
Published Date 1989-08
Publication Title Acta Medica Okayama
Volume volume43
Issue issue4
Publisher Okayama University Medical School
Start Page 197
End Page 202
ISSN 0386-300X
NCID AA00508441
Content Type Journal Article
language 英語
File Version publisher
Refereed True
PubMed ID 2678902
Web of Science KeyUT A1989AP79100001