Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin.
A photo-activatable analogue of biotin, N-(4-azido-2-nitrophenyl)-N'-(N-d-biotinyl-3-aminopropyl)-N'-methyl-1,3- propanediamine (photobiotin), has been synthesized and used for the rapid and reliable preparation of large amounts of stable, non-radioactive, biotin-labelled DNA and RNA hybridization probes. Upon brief irradiation with visible light, photobiotin formed stable linkages with single- and double-stranded nucleic acids yielding probes which were purified from excess reagent by 2-butanol extraction and ethanol precipitation. Using single-stranded phage M13 DNA probes chemically labelled with one biotin per 100-400 residues and dot-blot hybridization reactions on nitrocellulose, as little as 0.5 pg (6 X 10(-18) mol) of target DNA was detected colorimetrically by avidin or streptavidin complexes with acid or alkaline phosphatase from three commercial sources. The sensitivity of detection of target RNA in dot-blots and Northern blots was equivalent to that obtained with 32p-labelled DNA probes. Photobiotin was also used for the labelling of proteins with biotin.
Forster
A C
AC
McInnes
J L
JL
Skingle
D C
DC
Symons
R H
RH
D001386:Azides / Q000138:chemical synthesis / Q000378:metabolism*; D001710:Biotin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D003124:Colorimetry; D004247:DNA / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000378:metabolism*; D013053:Spectrophotometry
D001386:Azides; D012313:RNA; D001710:Biotin; D004247:DNA; C045549:photobiotin