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.
Kennedy
K E
KE
Department of Biology, East Carolina University, Greenville, NC 27858.
Daskalakis
S A
SA
Davies
L
L
Zwadyk
P
P
D001426:Bacterial Proteins; D001710:Biotin / Q000276:immunology; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis*; D006090:Gram-Negative Bacteria / Q000235:genetics / Q000302:isolation & purification; D009693:Nucleic Acid Hybridization; D019809:Streptavidin; D013450:Sulfones / Q000276:immunology; D001618:beta-Lactamases / Q000235:genetics
D001426:Bacterial Proteins; D015342:DNA Probes; D004269:DNA, Bacterial; D013450:Sulfones; D001710:Biotin; D019809:Streptavidin; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1