Non-radioactive hybridization probes prepared using M13 phage vector and the universal sequencing primer.
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.
Ikeda
S
S
Department of Biochemistry, Cancer Institute, Okayama University Medical School, Japan.
Yamamoto
M
M
Nagao
K
K
Zhang
B
B
Watanabe
S
S
Oda
T
T
D001435:Bacteriophages / Q000235:genetics*; D001710:Biotin; D003001:Cloning, Molecular; D015342:DNA Probes; D004279:DNA, Viral / Q000235:genetics*; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; D005822:Genetic Vectors; D006801:Humans; D009693:Nucleic Acid Hybridization; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019908:Proto-Oncogene Proteins c-raf; D011519:Proto-Oncogenes; D015183:Restriction Mapping; D013698:Templates, Genetic
D015342:DNA Probes; D004279:DNA, Viral; D011518:Proto-Oncogene Proteins; D001710:Biotin; D019908:Proto-Oncogene Proteins c-raf; D015246:Deoxyribonuclease EcoRI; C046545:CCWGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific