Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots.
Biotin-labelled DNA probes, prepared by nick-translation in the presence of biotinylated analogs of TTP, are hybridized to DNA or RNA immobilized on nitrocellulose filters. After removal of residual probe, the filters are incubated for 2--5 min with a preformed complex made with avidin-DH (or streptavidin) and biotinylated polymers of intestinal alkaline phosphatase. The filters are then incubated with a mixture of 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium, which results in the deposition of a purple precipitate at the sites of hybridization. This procedure will detect target sequences in the 1- to 10-pg range after enzyme incubation periods of 1 hr or less. The incubation period can be extended up to 24 hr, if required, to increase the color intensity of the hybridization signal. Furthermore, at high probe concentrations (250--7560 ng/ml), biotin-labeled DNA exhibits lower nonspecific binding to nitrocellulose than does radiolabeled DNA, so hybridization times required for the analysis of unique mammalian gene sequences can be decreased to 1--2 hr. This nonradiographic method of probe detection should be of general utility for genetic studies using Southern, RNA, or dot-blot hybridization protocols.
D000469:Alkaline Phosphatase; D000818:Animals; D001360:Avidin; D001710:Biotin; D002417:Cattle; D003101:Collodion; D003124:Colorimetry / Q000379:methods; D004247:DNA / Q000032:analysis*; D005260:Female; D005914:Globins / Q000235:genetics; D006801:Humans; D007202:Indicators and Reagents; D009693:Nucleic Acid Hybridization; D010920:Placenta; D011247:Pregnancy; D012313:RNA
D007202:Indicators and Reagents; D001360:Avidin; D012313:RNA; D001710:Biotin; D005914:Globins; D003101:Collodion; D004247:DNA; D000469:Alkaline Phosphatase