Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots.

Abstract

Biotin-labeled DNA probes, prepared by nicktranslation 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 periodwof 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 (250-750 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.Previous reports from this laboratory described the synthesis of biotin-labeled analogs of TTP and UTP that can be enzymatically incorporated into DNA and RNA, respectively (1, 2). The resulting biotin-labeled polynucleotides exhibit reassociation kinetics similar to those of biotin-free polymers and they function effectively as hybridization probes in situ. Hybridization signals can be visualized by indirect immunofluorescence, immunoperoxidase, or immuno-colloidal gold techniques, after incubation with a primary antibiotin antibody, and by cytochemical methods that use complexes of avidin and biotinylated peroxidase to detect the biotin-labeled probe. Such procedures have been applied successfully to the localization of specific sequences in Drosophila chromosomes (3, 4), mammalian metaphase chromosomes (5, 6), cultured cells (2, 7), and formalin-fixed tissue sections (2).However, none of the visualization methods used in these studies were able to detect sequences present at the level of one copy per mammalian cell. It was apparent, therefore, that the routine application of biotin-labeled probes in genetic analysis would require the development of more sensitive biotindetection systems.Here we report the synthesis of biotinylated polymers of alkaline phosphatase and the construction of complexes of avidin (or streptavidin) and enzyme polymer that are 20-to 50-fold more sensitive than immunologic or affinity reagents used previously. We also describe a rapid and sensitive procedure for visualizing biotin-labeled DNA probes after hybridization to DNA or RNA immobilized on nitrocellulose filters.

Cloning and Functional Expression of a Mammalian Gene for a Peroxisomal Sarcosine Oxidase

“…The procedure for affinity purification followed the protocol of Höhfeld et al (31). Detection was performed with a 1:15 000 dilution of alkaline phosphatase-coupled goat anti-Ig from rabbit (Sigma) in a nitro blue tetrazolium/4-bromo-5-chloro-3-indolyl phosphate-coupled reaction, as described (32). Alternatively, the enhanced chemiluminescence kit (Amersham, Braunschweig, Germany) was used for immunodetection.…”

Fluorometric Deoxyribonucleic Acid-Deoxyribonucleic Acid Hybridization in Microdilution Wells as an Alternative to Membrane Filter Hybridization in which Radioisotopes Are Used To Determine Genetic Relatedness among Bacterial Strains

“…Biotinylated DNA has been used for Southern and dot blot hybridizations (10,14). Hybridized DNA fragments are usually visualized with alkaline phosphatase or peroxidase color detection methods (10,14,17). We used photobiotinylated DNA for quantitative detection of DNA-DNA hybridization to determine genetic relatedness among microorganism for taxonomic studies (4, 5,19).…”

FastTag™ Nucleic Acid Labeling System: A Versatile Method for Incorporating Haptens, Fluorochromes and Affinity Ligands into DNA, RNA and Oligonucleotides

“…Recently, there has been an increasing shift from radioactive to nonradioactive nucleic acid probes (5,6) because of greater experimental flexibility afforded by nonradioactive labels and the hazards associated with radioactivity. However, many nonradioactive systems suffer from a drawback common to the radioactive methods in that they require the use of costly enzymes and labeled (deoxy)ribonucleotides.…”