An alternative nonradioactive method for labeling DNA using biotin.
An alternative nonradioactive labeling method and a highly sensitive technique for detecting specific DNA sequences are described. The labeling method requires the "Klenow" fragment of DNA polymerase I and random hexanucleotides (synthesized or naturally extracted) as a primer for the production of highly sensitive DNA probes. The system has three main steps: (i) labeling of DNA with biotinylated 11-dUTP; (ii) detection of biotinylated DNA by a one-step procedure with streptavidin-alkaline phosphatase complex; (iii) blocking of background with Tween 20. Twenty attograms (2 X 10(-17) g) of pBR322 plasmid DNA was detected by dot-blot hybridization. Upon Southern blot hybridization, 7.4 fg (7.4 X 10(-15) g) of pBR322 HindIII DNA was detected using the biotinylated pBR322 plasmid DNA probe; 40.8 ag and 7.4 fg of lambda HindIII DNA were detected with the biotinylated whole lambda DNA probe by dot and Southern blot hybridization, respectively. Specific bands were also detected with the biotinylated argininosuccinolyase probe upon Northern blotting of mouse poly(A+) RNA. Further applications for in situ hybridization are also described.
Takahashi
T
T
Department of Bacteriology, Yokohama City University School of Medicine, Japan.
Mitsuda
T
T
Okuda
K
K
D000818:Animals; D010582:Bacteriophage lambda / Q000032:analysis; D001710:Biotin / Q000032:analysis*; D015152:Blotting, Northern; D004247:DNA / Q000032:analysis*; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis; D006801:Humans; D051379:Mice; D009693:Nucleic Acid Hybridization; D010761:Phosphorus Radioisotopes; D012333:RNA, Messenger / Q000032:analysis
D015342:DNA Probes; D004279:DNA, Viral; D010761:Phosphorus Radioisotopes; D012333:RNA, Messenger; D001710:Biotin; D004247:DNA