Abstract
A nonradioactive labelling and detection method for plant genomic DNA analysis is compared to the radioactive method. The radioisotopes are replaced by a nucleotide, digoxigenin-11-dUTP, and the signal detection is accomplished by the enzymatic reaction of alkaline phosphatase, conjugated to anti-digoxigenin antibodies, with the chemiluminescent substrate AMPPD (3-(2′-spiroadamantane)-4-methoxy-4(3″ phosphorytoxy) phenyl-1, 2-dioxetane). The sensitivity of the radioactive and nonradioactive methods are directly compared using identical Southern blots subjected to the radioactive and nonradioactive detection. The advantages of this nonradioactive method are discussed.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Allefs, I.I.H.M., Salentijn, E.M.I., Krens, F.A. and Rouwendal, G.I.A. (1990) Optimization of non-radioactive Southern blot hybridization: single copy detection and reuse of blots.Nucl. Acids Res. 18, 3099–100.
Bronstein, I., Edwards, B. and Voyta, I.C. (1989a) 1,2-Dioxetanes: novel chemiluminescent enzyme substrates. Application to immunoassays.J. Biolumin. Chemilumin. 4, 99–111.
Bronstein, I., Voyta, I.C. and Edwards, B. (1989b) A comparison of chemiluminescent and colorimetric substrates in a hepatitis B virus DNA hybridization assay.Anal. Biochem. 180, 95–8.
Bronstein, I. and McGrath, P. (1989) Chemiluminescence lights up.Nature 338, 599–600.
Bronstein, I. and Kricka, L.I. (1989) Clinical applications of luminescent assays for enzymes and labels.J. Clinical Lab. Anal. 3, 316–22.
Bronstein, I., Voyta, I.C., Lazzari, K.G., Murphy, O., Edwards, B. and Kricka, L.I. (1990) Rapid and sensitive detection of DNA in Southern blots with chemiluminescence.Bio-Techniques 8, 310–4.
Beck, S., O'Keeffe, T., Coull, M.I., and Köster, H. (1989) Chemiluminescent detection of DNA: application for DNA sequencing and hybridization.Nucl. Acids Res. 17, 5115–23.
Beck, S. and Köster, H. (1990) Applications of Dioxetane chemiluminescent probes to molecular biology.Anal. Chem. 62, 2258–70.
Cate, R.L., Ehrenfels, Ch.W., Wysk, M., Tizard, R., Voyta, I.C., Murphy, O.J., III, and Bronstein, I. (1991) Genomic Southern analysis with alkaline-phosphatase-conjugated oligonucleotide probes and the chemiluminescent substrate AMPPD.Genet. Anal. Tech. Appl. 8, 102–6.
Düring, K. (1991) Ultrasensitive chemiluminescent and colorigenic detection of DNA, RNA and proteins in plant molecular biology:Anal. Biochem. 196, 433–8.
Feinberg, A.P. and Vogelstein, B. (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity.Anal. Biochem. 132, 6–13.
Höltke, H.I., Sagner, G., Kessler, Ch. and Schmitz G. (1992) Sensitive chemiluminescent detection of Digoxigenin-labelled nucleic acids: a fast and simple protocol and its application.Bio Techniques 12, 104–13.
Ishii, T., Panaud, O., Brar, D.S. and Khush, G.S. (1990) Use of non-radioactive digoxigenin-labelled DNA probes for RFLP analysis in rice.Pl. Mol. Biol. Rep. 8, 167–71.
Kreike, C.M., de Koning, I.R.A. and Krens, F.A. (1990) Non-radioactive detection of single-copy DNA-DNA hybrids.Pl. Mol. Biol. Rep. 8, 172–9.
Karcher, S.I. and Goodner, B.W. (1991) Chemiluminescent detection of multiple copy genes.Focus 12, 110–1.
Lanzillo, I.I. (1991) Chemiluminescent nucleic acid detection with digoxigenin-labelled probes: a model system with probes for angiotensin converting enzyme which detect less than one attomole of target DNA.Anal. Biochem. 194, 45–53.
Maniatis, T., Fritsch, E.F. and Sambrook, I. (1982)Molecular Cloning: a Laboratory Manual, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Murray, M.G. and Thompson, W.F. (1980) Rapid isolation of high molecular weight plant DNA.Nucl. Acids Res. 8, 4321–5.
Peterhans, A., Schlüpmann, H., Basse, Ch. and Paszkowsky, J. (1990) Intrachromosomal recombination in plants.EMBO J. 9, 3437–5.
Pollard-Knight, D. (1990) Current methods in nonradioactive nucleic acid labelling and detection.Technique 3, 113–32.
Schnorf, M., Neuhaus-Url, G., Galli, A., Iida, S., Potrykus, I. and Neuhaus, G. (1991) An improved approach for transformation of plant cells by microinjection: molecular and genetic analysis.Transgenic Res. 1, 23–30.
Tizard, R., Cate, L.R., Ramachandran, K.L., Wysk, M., Voyta, I.C., Murphy, O.I., and Bronstein, I. (1990) Imaging of DNA sequences with chemiluminescence.Proc. Natl Acad. Sci. USA 87, 4514–8.
Weising, K., Beyermann, B., Ramser, J. and Kahl, G. (1991) Plant DNA fingerprinting with radioactive and digoxigenated oligonucleotide probes complementary to simple repetitive DNA sequeces.Electrophoresis 12, 159–69.
Zachar, V., Mayer, V., Aboagye-Mathiesen, G., Norskov-Lauritsen, N. and Ebbesen, P. (1991) Enhanced chemiluminescence-based hybridization analysis for PCR-mediated HIV-1 DNA detection offers an alternative to32P-labelled probes.J. Virol. Meth. 33, 391–5.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Neuhaus-Url, G., Neuhaus, G. The use of the nonradioactive digoxigenin chemiluminescent technology for plant genomic Southern blot hybridization: A comparison with radioactivity. Transgenic Research 2, 115–120 (1993). https://doi.org/10.1007/BF01969385
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01969385