Abstract
Checkerboard DNA-DNA hybridization (CKB) is a technique that provides a simultaneous quantitative analysis of 40 microbial species against up to 28 mixed microbiota samples on a single membrane; using digoxigenin (DIG)-labeled, whole-genome DNA probes. Developed initially to study the predominantly Gram-negative dental plaque microorganisms involved in periodontitis, we modified the probe species composition to focus on putative pathogens involved in the development of dental caries. CKB analysis is applicable to species from other biodiverse ecosystems and to a large number of samples. The major limitations are that high-quality DNA is required for the preparation of DIG-labeled probes and standards, and that probe specificity requires careful evaluation. Overall, CKB analysis provides a powerful ecological fingerprint of highly biodiverse microbiota based on key cultivable bacteria.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ward, B. B. (2002) How many species of procaryotes are there? Proc. Nat. Acad. Sci. USA 99, 10,234–10,236.
Paster, B. J., Boches, S. K., Galvin, J. L., et al. (2001) Bacterial diversity in human subgingival plaque. J. Bacteriol. 183, 3770–3783.
Becker, M. R., Paster, B. J., Leys, E. J., et al. (2002) Molecular analysis of bacterial species associated with childhood caries. J. Clin. Microbiol. 40, 1001–1009.
Brinig, M. M., Lepp, P. W., Ouverney, C. C., Armitage, G. C., and Relman, D. A. (2003) Prevalence of bacteria of division TM7 in human subgingival plaque and their asociation with disease. Appl. Environ. Microbiol. 69, 1687–1694.
Harper-Owen, R., Dymock, D., Booth, V., Weightman, A. J., and Wade, W. G. (1999) Detection of unculturable bacteria in periodontal health and disease by PCR. J. Clin. Microbiol. 37, 1469–1473.
Torsvik, V., Øveras, L., and Thingstad, T. (2002) Prokaryotic diversity—magnitude, dynamics and controlling factors. Science 296, 1064–1066.
Osborn, A. M., Moore, E. R. B., and Timmis, K. N. (2000) An evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure and dynamics. Environ. Microbiol. 2, 39–50.
Muyzer, G., De Wall, E. C., and Unterlinden, A. G. (1993) Profiling of complex microbial population by denaturing gradient gel electrophoresis of polymerase chain reaction-amplified genes coding for 16S RNA. Appl. Environ. Microbiol. 59, 695–700.
Munson, M. A., Bannerjee, A., Watson, T. F., and Wade, W. G. (2004) Molecular analysis of the microflora associated with dental caries. J. Clin. Microbiol. 42, 3023–3029.
Anderson, S. A., Sissons, C. H., Coleman, M. J., and Wong, L. (2002) Application of carbon-source utilisation patterns to measue the metabolic similarity of complex dental plaque biofilm microcosms. Appl. Environ. Microbiol. 68, 5779–5783.
Roche Applied Science. (2000) The DIG Application Manual for Filter Hybridization. Available at: http://www.roche-applied-science.com/.
Socransky, S. S., Smith, C., Martin, L., Paster, B. J., Dewhirst, F. E., and Levin, A. E. (1994) “Checkerboard” DNA-DNA hybridization. Biotechniques 17, 788–792.
Socransky, S. S., Haffajee, A. D., Cugini, M. A., Smith, C., and Kent, R. L. (1998) Microbial complexes in subgingival plaque. J. Clin. Periodontol. 25, 134–144.
Socransky, S. S., Haffajee, A. D., Smith, C., et al. (2004) The use of checkerboard DNA:DNA hybridization to study complex microbial ecosystems. Oral Microbiol. Immunol. 19, 352–362.
Socransky, S. S. and Haffajee, A. D. (2002) Dental biofilms: difficult therapeutic targets. Periodontology 2000 28, 12–55.
Haffajee, A. D., Socransky, S. S., Feres, M., and Ximenez-Fyvie, L. A. (1999) Plaque microbiology in health and disease, in Dental Plaque Revisited (Newman, H. N. and Wilson, M., eds.), Bioline, Cardiff, UK, pp. 255–282.
Wall-Manning, G. and Sissons, C. H. (2002) The effect of regrowth after treatment with antimicrobial on the species composition of dental plaque microcosms, in New Zealand Microbiological Society Conference, Christchurch, New Zealand, p. 156.
Sissons, C. H., Wong, L., and An, Y. H. (2000) Culture and analysis of bacterial biofilms, in Handbook for Studying Bacterial Adhesion (An, Y. H. and Friedman, R. J., eds.), Humana, Totowa, NJ, pp. 133–169.
Sissons, C. H. (1997) Artificial dental plaque biofilm model systems. Adv. Dent. Res. 11, 110–126.
Sissons, C. H., Cutress, T. W., Hoffman, M. P., and Wakefield, J. S. J. (1991) A multi-station dental plaque microcosm (artificial mouth) for the study of plaque growth, metabolism, pH and mineralisation. J. Dent. Res. 70, 1409–1416.
Tanner, A., Maiden, M. F. J., Macuch, P. J., Murray, L. L., and Kent, R. L. (1998) Microbiota of health, gingivitis and initial periodontitis. J. Clin. Periodontol. 25, 134–144.
Smith, G. L. F., Sockransky, S. S., and Smith, C. M. (1989) Rapid method for the purification of DNA from subgingival organisms. Oral Microbiol. Immunol. 4, 47–51.
Wall-Manning, G. M., Sissons, C. H., Anderson, S. A., and Lee, M. (2002) Checkerboard DNA-DNA hybridisation technology focussed on the analysis of Gram-positive cariogenic bacteria. J. Microbiol. Meth. 51, 301–311.
Ludwig, J. A. and Reynolds, J. F. (eds.) (1988) Statistical Ecology, John Wiley and Sons, New York.
Ximenez-Fyvie, L. A., Haffajee, A. D., Martin, L., Tanner, A., Macuch, P., and Socransky, S. S. (1999) Identification of oral Actinomyces species using DNA probes. Oral Microbiol. Immunol. 14, 257–265.
Bjournson, A. J. and Cooper, J. E. (1996) Subtraction hybridization for the isolation of strain-specific Rhizobium DNA probes. Meth. Mol. Biol. 50, 145–154.
Janda, J. M. and Abbott, S. A. (2002) Bacterial identification for publication: when is enough enough? J. Clin. Microbiol. 46, 1887–1891.
Paster, B. J., Bartoszyk, I. M., and Dewhirst, F. E. (1998) Identification of oral streptococci using PCR-based reverse-capture, checkerboard hybridization. Meth. Cell Sci. 20, 223–231.
Socransky, S. S., Haffajee, A. D., Smith, C., Martin, L., Haffajee, J. A., Uzel, N. G., and Goodson, J. M. (2004) Use of Checkerboard DNA-DNA hybridization to study complex microbial ecosystems. Oral Microbiol. Immunol. 19, 352–362.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc.
About this protocol
Cite this protocol
Gellen, L.S., Wall-Manning, G.M., Sissons, C.H. (2007). Checkerboard DNA-DNA Hybridization Technology Using Digoxigenin Detection. In: Hilario, E., Mackay, J. (eds) Protocols for Nucleic Acid Analysis by Nonradioactive Probes. Methods in Molecular Biology, vol 353. Humana Press. https://doi.org/10.1385/1-59745-229-7:39
Download citation
DOI: https://doi.org/10.1385/1-59745-229-7:39
Publisher Name: Humana Press
Print ISBN: 978-1-58829-430-2
Online ISBN: 978-1-59745-229-8
eBook Packages: Springer Protocols