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Improvements of the membrane filter method for DNA:rRNA hybridization

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Abstract

We describe and recommend the following improvements of DNA:rRNA membrane filter hybridization methods. One of our aims was to avoid DNA release from filter discs during hybridization.

  1. 1.

    Our hybridization conditions are 2 SSC in aq. dest., with 20% formamide, 50 C, overnight for 16 hr.

  2. 2.

    Duplexing is over in 8–10 hr.

  3. 3.

    Formamide has to be very pure (O.D.≤0.2/cm light path at 270 nm).

  4. 4.

    RNAase treatment: 250 μg/5 ml 2 SSC/filter at 37 C for 1 hr.

  5. 5.

    Our conditions for stepwise thermal denaturation are: 5°C steps from 50C to 90C in 1.5 SSC in 20% formamide.

  6. 6.

    Single-stranded DNA, fixed on membrane filters, and stored in vacuo at 4C, can be used reliably for hybridization for up to 20 months.

  7. 7.

    Concentrated DNA in 0.1 SSC, quick-frozen at −50 C and stored at −90 C for up to 2 years can be used for hybridization without much change.

  8. 8.

    A CsCl gradient purification step yields much purer DNA, but increases the release of DNA from filters by about 20%. Filters with 20% more DNA is a compensation.

  9. 9.

    rRNA can be stored for 20 months in SSC or 2 SSC at −12C without changing the hybridization results.

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References

  • Bishop, J. O. 1970. Examination of an equilibrium interpretation of deoxyribonucleic acid-ribonucleic acid hybridization data.—Biochem. J.116: 223–228.

    PubMed  CAS  Google Scholar 

  • Bishop, J. O., Robertson, F. W., Burns, J. A. andMelli, M. 1969. Methods for the analysis of deoxyribonucleic acid-ribonucleic acid hybridization data.—Biochem. J.115: 361–370.

    PubMed  CAS  Google Scholar 

  • Bonner, J., Kung, G. andBekhor, I. 1967. A method for the hybridization of nucleic acid molecules at low temperature.—Biochemistry6: 3650–3653.

    Article  PubMed  CAS  Google Scholar 

  • Burton, K. 1956. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid.—Biochem. J.62: 315–323.

    PubMed  CAS  Google Scholar 

  • Crombach, W. H. J. 1972. DNA base composition of soil arthrobacters and other coryneforms from cheese and sea fish.—Anttonie van Leeuwenhoek38: 105–120.

    Article  CAS  Google Scholar 

  • Crombach, W. H. J. 1973. Deep-freezing of bacterial DNA for thermal denaturation and hybridization experiments.—Antonie van Leeuwenhoek39: 249–255.

    PubMed  CAS  Google Scholar 

  • Daniel, J. C., Greene, R. F., Mitchell, R. A. andFlickinger, R. A. 1970. An improved method of detecting DNA-RNA hybrids bound to nitrocellulose filters.—Anal. Biochem.37: 330–332.

    Article  PubMed  CAS  Google Scholar 

  • De Ley, J. 1971. Hybridization of DNA, p. 311–329.In Norris, J. R. and Ribbons, D. W., (eds.), Methods in Microbiology Vol. 5A.—Academic Press, London and New York.

    Google Scholar 

  • De Ley, J., Cattoir, H. andReynaerts, A. 1970. The quantitative measurement of DNA hybridization from renaturation rates.—Eur. J. Biochem.12: 133–142.

    Article  PubMed  Google Scholar 

  • De Ley, J. andFriedman, S. 1964. Deoxyribonucleic acid hybrids of acetic acid bacteria. —J. Bacteriol.88: 937–945.

    Google Scholar 

  • De Ley, J., Park, I. W., Tijtgat, R. andVan Ermengem, J. 1966. DNA homology and taxonomy ofPseudomonas andXanthomonas.—J. Gen. Microbiol.42: 43–56.

    PubMed  Google Scholar 

  • De Ley, J. andTijtgat, R. 1970. Evaluation of membrane filter methods for DNA-DNA hybridizations.—Antonie van Leeuwenhoek36: 461–474.

    Article  PubMed  Google Scholar 

  • Fry, M. andArtman, M. 1969. Deoxyribonucleic acid-ribonucleic acid hybridizations. —Biochem. J.115: 287–294.

    PubMed  CAS  Google Scholar 

  • Gillespie, D. 1968. The formation and detection of DNA: RNA hybrids, p. 641–668.In Colowick, S.P. and Kaplan, N.O., (Eds.), Methods in Enzymology, Vol. XII B.—Academic Press, New York and London.

    Google Scholar 

  • Gillespie, S. andGillespie, D. 1971. Ribonucleic acid-deoxyribonucleic acid hybridization in aqueous solutions and in solutions containing formamide.—Biochem. J.125: 481–487.

    PubMed  CAS  Google Scholar 

  • Gillespie, D. andSpiegelman, S. 1965. A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane.—J. Mol. Biol.12: 829–842.

    PubMed  CAS  Google Scholar 

  • Gillis, M., De Ley, J. andDe Cleene, M. 1970. The determination of molecular weight of bacterial genome DNA from renaturation rates.—Eur. J. Biochem.12: 143–153.

    Article  PubMed  CAS  Google Scholar 

  • Grienenberger, J. M. etSimon, D. 1972. Etude de la structure et de la dégradation des acides ribonucléiques ribosomaux de quelques bactéries du genreAgrobacterium.—C.R. Acad. Sci. (Paris)274: 1399–1402.

    CAS  Google Scholar 

  • Hayashi, M. andSpiegelman, S. 1961. The selective synthesis of informational RNA in bacteria.—Proc. Nat. Acad. Sci. U.S.A.47: 1564–1580.

    Article  CAS  Google Scholar 

  • Heberlein, G. T., De Ley, J. andTytgat, R. 1967. Deoxyribonucleic acid homology and taxonomy ofAgrobacterium, Rhizobium andChromobacterium.—J. Bacteriol.94: 116–124.

    PubMed  CAS  Google Scholar 

  • Kirby, K. S. 1957. A new method for the isolation of deoxyribonucleic acids: evidence on the nature of bonds between deoxyribonucleic acid and protein.—Biochem. J.66: 495–504.

    PubMed  CAS  Google Scholar 

  • Kirby, K. S., Fox-Carter, E. andGuest, M. 1967. Isolation of deoxyribonucleic acid and ribosomal ribonucleic acid from bacteria.—Biochem. J.104: 258–262.

    PubMed  CAS  Google Scholar 

  • Kourilsky, Ph., Manteuil, S., Zamansky, M. H. andGros, F. 1970. DNA-RNA hybridization at low temperature in the presence of urea.—Biochem. Biophys. Res. Comm.41: 1080–1087.

    Article  PubMed  CAS  Google Scholar 

  • Le Goff, L. 1968. Acides ribonucléiques des bactéries du crown-gallAgrobacterium tumefaciens (Smith et Town) Conn. exposées au borate de sodium.—Ann. Inst. Pasteur115: 232–248.

    Google Scholar 

  • Lochhead, A. G. andBurton, M. O. 1954. Qualitative studies of soil microorganisms. XII. Characteristics of vitamin-B12-requiring bacteria.—Can. J. Microbiol.1: 319–330.

    Article  Google Scholar 

  • Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acid from microorganisms. —J. Mol. Biol.3: 208–218.

    Article  CAS  Google Scholar 

  • McConaughy, B. L., Laird, C. D. andMcCarthy, B. J. 1969. Nucleic acid reassociation in formamide.—Biochemistry8: 3289–3295.

    Article  PubMed  CAS  Google Scholar 

  • McConkey, E. H. 1968. The fractionation of RNA's by sucrose gradient centrifugation, p. 620–634.In S. P. Colowick and N. O. Kaplan, (Eds), Methods in Enzymology, Vol. XII. —Academic Press, New York and London.

    Google Scholar 

  • Melli, M., Whitfield, C., Rao, K. V., Richardson, M. andBishop, J. O. 1971. DNA-RNA hybridization in vast DNA excess.—Nature New Biol.231: 8–12.

    PubMed  CAS  Google Scholar 

  • Meijs, W. H. andSchilperoort, R. A. 1971. Determination of the amount of DNA on nitrocellulose membrane filters.—FEBS Letter12: 166–168.

    Article  CAS  Google Scholar 

  • Midgley, J. E. M. 1965. Effects of different extraction procedures on the molecular characteristics of bacterial ribosomal ribonucleic acid.—Biochim. Biophys. Acta95: 232–243.

    PubMed  CAS  Google Scholar 

  • Moore, R. L. andMcCarthy, B. J. 1967. Comparative study of ribosomal ribonucleic acid cistrons in Enterobacteria and Myxobacteria.—J. Bacteriol.94: 1066–1074.

    PubMed  CAS  Google Scholar 

  • Noll, H. andStutz, E. 1968. The use of sodium and lithium dodecyl sulfate in nucleic acid isolation, p. 129–155.In S. P. Colowick and N. O. Kaplan, (eds.), Methods in Enzymology, Vol. XIIB.—Academic Press, New York and London.

    Google Scholar 

  • Nygaard, A. P. andHall, B. D. 1963. A method for the detection of RNA-DNA complexes. —Biochem. Biophys. Res. Comm.12: 98–104.

    Article  PubMed  CAS  Google Scholar 

  • Nygaard, A. P. andHall, B. D. 1964. Formation and properties of RNA-DNA complexes. —J. Mol. Biol.9: 125–142.

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Laboratory for Microbiology and Microbial Genetics, Faculty of Sciences, State University, K. L. Ledeganckstraat 35, B-9000, Gent, Belgium

    J. De Ley & J. De Smedt

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  1. J. De Ley
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  2. J. De Smedt
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De Ley, J., De Smedt, J. Improvements of the membrane filter method for DNA:rRNA hybridization. Antonie van Leeuwenhoek 41, 287–307 (1975). https://doi.org/10.1007/BF02565064

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  • DOI: https://doi.org/10.1007/BF02565064

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