Advertisement

Biologia

, Volume 67, Issue 4, pp 644–648 | Cite as

Isolation and purification of microbial community DNA from soil naturally enriched for chitin

  • Pullabhotla V. S. R. N. Sarma
  • Vadlamudi Srinivas
  • Kondreddy Anil
  • Appa Rao PodileEmail author
Full Paper Section Cellular and Molecular Biology
  • 263 Downloads

Abstract

We made an attempt to isolate and purify metagenomic DNA from chitin enriched soil. In this communication we report a modified direct lysis method for soil DNA extraction including initial pre-lysis washing of sample, followed by a rapid polyvinylpyrrolidone-agarose-based purification and electroelution of DNA using Gene-capsule™ assembly. Rapidity was achieved using low molarity conducting media (sodium-borate buffer) for electrophoresis by reducing run time for both the gel electrophoresis and electroelution. Extracted DNA was sufficiently pure and of high quality, evidenced by amplification of 16S rDNA and chitinase genes by PCR. Metagenomic nature of the DNA was confirmed by running V3 (16S rDNA) region amplicons using denaturing gradient gel electrophoresis. This method requires 30 min for purification, and less than 2 h for complete execution of protocol and becomes the first report on the isolation of metagenomic DNA from soil naturally enriched for chitin.

Key words

metagenomic DNA DNA purification chitin-enriched soil denaturing gradient gel electrophoresis 

Abbreviations

chitinase A

chi-A

DGGE

denaturing gradient gel electrophoresis

MC

Mahtani Chitosan

PVP

polyvinylpyrrolidone

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Brody J.R., Calhoun E.S., Gallmeier E., Creavalle T.D. & Kern S.E. 2004. Ultra-fast high-resolution agarose electrophoresis of DNA and RNA using low-molarity conductive media. BioTechniques 37: 598–602.PubMedGoogle Scholar
  2. Cottrell M.T., Moore J.A. & Kirchman D.L. 1999. Chitinases from uncultured marine microorganisms. Appl. Environ. Microbiol. 65: 2553–2557.PubMedGoogle Scholar
  3. Das S.N., Sarma P.V.S.R.N., Neeraja C., Malati N. & Podile A.R. 2010. Members of γ-Proteobacteria and Bacilli represent the culturable diversity of chitinolytic bacteria in chitin-enriched soils. World J. Microbiol. Biotechnol. 26: 1875–1881.CrossRefGoogle Scholar
  4. Desai C. & Madamwar D. 2007. Extraction of inhibitor-free metagenomic DNA from polluted sediments, compatible with molecular diversity analysis using adsorption and ion exchange treatments. Bioresour. Technol. 98: 761–768.PubMedCrossRefGoogle Scholar
  5. He J, Xu Z. & Hughes J. 2005. Prelysis washing improves DNA extraction from a forest soil. Soil Biol. Biochem. 37: 2337–2341.CrossRefGoogle Scholar
  6. Herrera R.R., Zapata J.N., Maya M.Z. & Martinez M.E.M. 2008. A simple silica based method for metagenomic DNA extraction from soil and sediments. Mol. Biotechnol. 40: 13–17.CrossRefGoogle Scholar
  7. Hobel C.F., Marteinsson V.T., Hreggvidsson G.O. & Kristjansson J.K. 2005. Investigation of the microbial ecology of intertidal hot springs by using diversity analysis of 16S rRNA and chitinases genes. Appl. Environ. Microbiol. 71: 2771–2776.PubMedCrossRefGoogle Scholar
  8. Ikeda S., Watanabe K.N., Minamisawa K. & Ytow N. 2004. Evaluation of soil DNA from arable land in Japan using a modified direct-extraction method. Microbes Environ. 19: 301–309.CrossRefGoogle Scholar
  9. Mabuchi N. & Araki Y. 2001. Cloning and sequencing of two genes encoding chitinases A and B from Bacillus cereus CH. Can. J. Microbiol. 47: 895–902.PubMedGoogle Scholar
  10. Muyzer G., de Waal E.C. & Uitterlinden A.G. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reactionamplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59: 695–700.PubMedGoogle Scholar
  11. Neeraja C, Moerschbacher B. & Podile A.R. 2010. Fusion of cellulose binding domain to the catalytic domain improves the activity and conformational stability of chitinase in Bacillus licheniformis DSM13. Bioresour. Technol. 101: 3635–3641.PubMedCrossRefGoogle Scholar
  12. Rajendhran J. & Gunasekaran P. 2008. Strategies for accessing soil metagenome for desiredapplications. Biotechnol. Adv. 26: 576–590.PubMedCrossRefGoogle Scholar
  13. Sambrook J. & Russel W.D. 2001. Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, New York, USA.Google Scholar
  14. Sato K., Azama Y., Nogawa M., Taguchi G. & Shimosaka M. 2010. Analysis of a change in bacterial community in different environments with addition of chitin or chitosan. J. Biosci. Bioeng. 109: 472–478.PubMedCrossRefGoogle Scholar
  15. Terahara T., Ikeda S., Noritake C., Minamisawa K., Ando K., Tsuneda S. & Harayama S. 2009. Molecular diversity of bacterial chitinases in arable soils and the effects of environmental factors on the chitinolytic bacterial community. Soil Biol. Biochem. 41: 473–480.CrossRefGoogle Scholar
  16. Tsai Y.L. & Olson B.H. 1991. Rapid method for direct extraction of DNA from soil and sediments. Appl. Environ. Microbiol. 57: 1070–1074.PubMedGoogle Scholar
  17. Xiao X., Yin X., Lin J., Sun L., You Z., Wang P. & Wang F. 2005. Chitinase genes in lake sediments if ardley island, Antarctica. Appl. Environ. Microbiol. 7: 7904–7909.CrossRefGoogle Scholar
  18. Yasir M, Aslam Z, Kim S.W., Lee S.W., Jeon C.O. & Chung Y.R. 2009. Bacterial community composition and chitinase gene diversity of vermicompost with antifungal activity. Bioresour. Technol. 100: 4396–4403.PubMedCrossRefGoogle Scholar
  19. Young C.C., Burghof R.L., Keim L.G., Minak-Bernero V., Lute J.R. & Hinton S.M. 1993. Polyvinylpyrrolidone-agarose gel electrophoresis purification of polymerase chain reactionamplifiable DNA from soils. Appl. Environ. Microbiol. 59: 1972–1974.PubMedGoogle Scholar
  20. Zhou J., Bruns M.A. & Tiedje J.M. 1996. DNA recovery from soils of diverse composition. Appl. Environ. Microbiol. 62: 316–322.PubMedGoogle Scholar

Copyright information

© Versita Warsaw and Springer-Verlag Wien 2012

Authors and Affiliations

  • Pullabhotla V. S. R. N. Sarma
    • 1
  • Vadlamudi Srinivas
    • 1
  • Kondreddy Anil
    • 1
  • Appa Rao Podile
    • 1
    Email author
  1. 1.Department of Plant Sciences, School of Life SciencesUniversity of HyderabadHyderabadIndia

Personalised recommendations