Nucleic Acids Research

This non-isotopic method for detection of nucleic acids is based on the in situ labelling of the nucleic acid by exposure to LW-irradiation. The different UW-induced photoproducts, mainly of the thymidine dimer type, are recognized by purified rabbit antibodies specific to the lesions introduced. The LWlabelled nucleic acids can then be visualized by conventional immunostaining procedures. A major advantage of the technique is the low cost and the ease by which the DNA is specifically labelled. The purified rabbit antibodies were shown to be specific for LW-irradiated DNA, and the method was applied for detection of specific DNA sequences hybridized to homologous target DNA on membrane support. We believe that the sensitivity of the method can be improved, and the significance of using different LW-doses, immunostaining methods and membrane types is discussed. INTRODUCTION There exist several non-isotopic methods for labelling of DNA. These methods circumvent most of the disadvantages associated with the use of radioisotopes ( e.g. short shelflife and safety precautions ), but they are usually less sensitive (1). The principle of some non-isotopic methods are enzymatic or chemical modification of DNA, followed by detection of modified DNA by antibodies. For example the nick-translation technique can be utilized for incorporation of biotinylated dUTP (2,3). The biotin-labelled DNA then can be detected by either enzyme conjugated avidin or streptavidin, by enzyme conjugated biotin specific antibodies, or by a complex of streptavidin and biotinylated enzymes (BluGENETM) However, the enzymatic incorporation of biotinylated nucleotides is costly and time consuming. Forster et al. (4) use photobiotin which 539 Nucleic Acids Research Volume 17 Number 2 1989 Nucleic Acids Research forms stable linkages with DNA upon irradiation with visible light (PhotobiotinTM). DNA can also be labelled by direct base modification, either by alkylation (5) or sulfonation (ChemiprobeTM) (6). We here describe a non-isotopic technique for labelling and detection of DNA. It is based on UV-irradiation as a means to label DNA, followed by detection of the UV-labelled DNA by immunostaining methods. A major advantage of the method resides in the very simple labelling of DNA by exposure to ultraviolet light for a few minutes. Such UV-irradiation generates several photoproducts which are stable and can be recognized by antibodies raised against UV-irradiated DNA. These antibodies have been shown to contain major specificities against photoproducts of the pyrimidine dimer type such as (64) photoproducts and cyclobutane thymine dimers (7, 8) . The bound UV-DNA specific antibodies can be visualized by different immunostaining methods. This paper describe the development of the method and its use for detection of UW-labelled probe DNA hybridized to homologous target DNA on membrane support. MATERIAL AND METHODS Chemicals Lambda DNA and the restriction enzymes HindIII, EcoRI and BstEII were from New England Biolabs. Calf thymus and salmon sperm DNA, methylated bovine serum albumin, ovalbumin, dextran sulfate, and Tween 20 were from Sigma. Formamide was from Fluka and Mixed Bed Resin AG 501.X8 from Bio-Rad. Horse radish peroxidase (HRP) conjugated anti-rabbit IgG was obtained from Dakopatts A/S, while alkaline phosphatase conjugated anti-rabbit IgG was from Cooper Biochemical. The substrate 3,3'-diaminobenzidine (DAB) was from Sigma, and nitroblue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3indolylphosphate (BCIP) were from BRL. Normal swine serum was from GIBCO. Irradiation of DNA Irradiation of DNA was performed at 254 nm using a Phillips TUV 15 W ultraviolet lamp with a dose rate of 10 W/m2 . DNA was