Non-isotopic hybridization assays for bacterial DNA samples

The background problem associated with the use of streptavidin in detecting biotin-labelled probes hybridized to DNA in crude bacterial extracts has been investigated. We have found that streptavidin binds specifically to a limited number of polypeptides which are difficult to remove by rapid extraction processes. Altering the hybridization and detection protocols results in a marked but not complete reduction of non-specific background in streptavidin-biotin assays. Complete elimination of non-specific background was achieved only when streptavidin was replaced with antibodies for the detection of biotinylated or sulphone-modified probes. The antibody-sulphone and streptavidin-biotin dot blot assays described here require 4.5-5 hours to perform and can detect DNA sequences in samples extracted from 2 x 10(7) cells or fewer.