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Preparation of labeled nucleic acids (nick translation and iodination) for DNA homology and rRNA hybridization experiments

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Abstract

Nick-translated DNA preparations may contain very short fragments, causing inconsistent DNA homology results. This appears to be due to extensive fragmentation of the high molecular weight DNA used in the labeling procedure. DNA preparations from organisms containing DNA of low mol% G+C content appear to be the most fragmented. The problem was circumvented by labeling these DNA preparations with125I. Sample preparation protocols have been developed for the routine125I labeling of DNA and rRNA from a wide range of organisms.

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Literature Cited

  1. Chan, H. C., Ruyechan, W. T., Wetmur, J. G. 1976.In vitro iodination of low complexity nucleic acids without chain scission. Biochemistry15:5487–5490.

    PubMed  Google Scholar 

  2. Chelm, B. K., Hallick, R. B. 1976. Changes in the expression of the chloroplast genomeEuglena gracilis during chloroplast development. Biochemistry15:593–599.

    PubMed  Google Scholar 

  3. Commerford, S. L. 1971. Iodination of nucleic acidsin vitro. Biochemistry11:1993–1999.

    Google Scholar 

  4. Crosa, J. H., Brenner, D. J., Falkow, S. 1973. Use of singlestrand-specific nuclease for analysis of bacterial and plasmid deoxyribonucleic acid homo- and heteroduplexes. Journal of Bacteriology115:904–911.

    PubMed  Google Scholar 

  5. Edelman, M. 1975. Purification of DNA by affinity chromatography: removal of polysaccharide contaminants. Analytical Biochemistry65:293–297.

    PubMed  Google Scholar 

  6. Franklin, R. M. 1966. Purification and properties of the replicatine intermediate of the RNA bacteriophage R17. Proceedings of the National Academy of Sciences55:1504–1511.

    Google Scholar 

  7. Glisin, V., Crkvenjakov, R., Byus, C. 1974. Ribonucleic acid isolation by cesium chloride centrifugation. Biochemistry13:2633–2637.

    PubMed  Google Scholar 

  8. Grimont, P. A. D., Popoff, M. Y., Grimont, F., Coynault, C., Lemelin, M. 1980. Reproducibility and correlation study of three deoxyribonucleic acid hybridization procedures. Current Microbiology4:325–330.

    Google Scholar 

  9. Holdeman, L. V., Moore, W. E. C., Churn, P. J., Johnson, J. L. 1982.Bacteroides oris andBacteroides buccae, new species from human periodontitis and other human infections. International Journal of Systematic Bacteriology32:125–131.

    Google Scholar 

  10. Johnson, J. L. 1981. Genetic characterization, pp. 450–472. In: Gerhardt, P. (ed.), Manual of methods for general bacteriology. American Society for Microbiology, Washington, DC.

    Google Scholar 

  11. Johnson, J. L., Francis, B. S. 1975. Taxonomy of the clostridia: ribosomal ribonucleic acid homologies among the species. Journal of General Microbiology88:229–244.

    PubMed  Google Scholar 

  12. Lachance, M.-A. 1980. Simple method for determination of deoxyribonucleic acid relatedness by thermal elution in hydroxyapatite microcolumns. International Journal of Systematic Bacteriology30:433–436.

    Google Scholar 

  13. Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acid from microorganisms. Journal of Molecular Biology3:208–218.

    Google Scholar 

  14. McDonell, M. W., Simon, M. N., Studier, F. W. 1977. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. Journal of Molecular Biology110:119–146.

    PubMed  Google Scholar 

  15. Orosz, J. M., Wetmur, J. G. 1974.In vitro iodination of DNA. Maximizing iodination while minimizing degradation; use of buoyant density shifts for DNA-DNA hybrid isolation. Biochemistry,13:5467–5473.

    PubMed  Google Scholar 

  16. Rigby, P. W. J., Dieckmann, M., Rhodes, C., Berg, P. 1977. Labeling deoxyribonucleic acid to high specific activityin vitro by nick translation with DNA polymerase I. Journal of Molecular Biology113:237–251.

    PubMed  Google Scholar 

  17. Tereba, A., McCarthy, B. J. 1973. Hybridization of125I-labeled ribonucleic acid. Biochemistry12:4675–4679.

    PubMed  Google Scholar 

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  1. Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, 24061, Blacksburg, Virginia, USA

    Ylva M. Selin, Barbara Harich & J. L. Johnson

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  1. Ylva M. Selin
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  2. Barbara Harich
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  3. J. L. Johnson
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Selin, Y.M., Harich, B. & Johnson, J.L. Preparation of labeled nucleic acids (nick translation and iodination) for DNA homology and rRNA hybridization experiments. Current Microbiology 8, 127–132 (1983). https://doi.org/10.1007/BF01566970

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