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.
Our hybridization conditions are 2 SSC in aq. dest., with 20% formamide, 50 C, overnight for 16 hr.
-
2.
Duplexing is over in 8–10 hr.
-
3.
Formamide has to be very pure (O.D.≤0.2/cm light path at 270 nm).
-
4.
RNAase treatment: 250 μg/5 ml 2 SSC/filter at 37 C for 1 hr.
-
5.
Our conditions for stepwise thermal denaturation are: 5°C steps from 50C to 90C in 1.5 SSC in 20% formamide.
-
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.
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.
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.
rRNA can be stored for 20 months in SSC or 2 SSC at −12C without changing the hybridization results.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Bishop, J. O. 1970. Examination of an equilibrium interpretation of deoxyribonucleic acid-ribonucleic acid hybridization data.—Biochem. J.116: 223–228.
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.
Bonner, J., Kung, G. andBekhor, I. 1967. A method for the hybridization of nucleic acid molecules at low temperature.—Biochemistry6: 3650–3653.
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.
Crombach, W. H. J. 1972. DNA base composition of soil arthrobacters and other coryneforms from cheese and sea fish.—Anttonie van Leeuwenhoek38: 105–120.
Crombach, W. H. J. 1973. Deep-freezing of bacterial DNA for thermal denaturation and hybridization experiments.—Antonie van Leeuwenhoek39: 249–255.
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.
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.
De Ley, J., Cattoir, H. andReynaerts, A. 1970. The quantitative measurement of DNA hybridization from renaturation rates.—Eur. J. Biochem.12: 133–142.
De Ley, J. andFriedman, S. 1964. Deoxyribonucleic acid hybrids of acetic acid bacteria. —J. Bacteriol.88: 937–945.
De Ley, J., Park, I. W., Tijtgat, R. andVan Ermengem, J. 1966. DNA homology and taxonomy ofPseudomonas andXanthomonas.—J. Gen. Microbiol.42: 43–56.
De Ley, J. andTijtgat, R. 1970. Evaluation of membrane filter methods for DNA-DNA hybridizations.—Antonie van Leeuwenhoek36: 461–474.
Fry, M. andArtman, M. 1969. Deoxyribonucleic acid-ribonucleic acid hybridizations. —Biochem. J.115: 287–294.
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.
Gillespie, S. andGillespie, D. 1971. Ribonucleic acid-deoxyribonucleic acid hybridization in aqueous solutions and in solutions containing formamide.—Biochem. J.125: 481–487.
Gillespie, D. andSpiegelman, S. 1965. A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane.—J. Mol. Biol.12: 829–842.
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.
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.
Hayashi, M. andSpiegelman, S. 1961. The selective synthesis of informational RNA in bacteria.—Proc. Nat. Acad. Sci. U.S.A.47: 1564–1580.
Heberlein, G. T., De Ley, J. andTytgat, R. 1967. Deoxyribonucleic acid homology and taxonomy ofAgrobacterium, Rhizobium andChromobacterium.—J. Bacteriol.94: 116–124.
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.
Kirby, K. S., Fox-Carter, E. andGuest, M. 1967. Isolation of deoxyribonucleic acid and ribosomal ribonucleic acid from bacteria.—Biochem. J.104: 258–262.
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.
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.
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.
Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acid from microorganisms. —J. Mol. Biol.3: 208–218.
McConaughy, B. L., Laird, C. D. andMcCarthy, B. J. 1969. Nucleic acid reassociation in formamide.—Biochemistry8: 3289–3295.
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.
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.
Meijs, W. H. andSchilperoort, R. A. 1971. Determination of the amount of DNA on nitrocellulose membrane filters.—FEBS Letter12: 166–168.
Midgley, J. E. M. 1965. Effects of different extraction procedures on the molecular characteristics of bacterial ribosomal ribonucleic acid.—Biochim. Biophys. Acta95: 232–243.
Moore, R. L. andMcCarthy, B. J. 1967. Comparative study of ribosomal ribonucleic acid cistrons in Enterobacteria and Myxobacteria.—J. Bacteriol.94: 1066–1074.
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.
Nygaard, A. P. andHall, B. D. 1963. A method for the detection of RNA-DNA complexes. —Biochem. Biophys. Res. Comm.12: 98–104.
Nygaard, A. P. andHall, B. D. 1964. Formation and properties of RNA-DNA complexes. —J. Mol. Biol.9: 125–142.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
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
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF02565064