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Purification of mitochondrial and plastid DNA

Nature Protocols volume 2, pages 652–660 (2007)Cite this article

Abstract

The size, structure and conformation of mitochondrial and plastid genomes differ dramatically among eukaryotes. Similarly, the yield and purity of extracted organelle DNA also vary, and are crucial factors for the success of restriction mapping and sequencing experiments. We describe here procedures for the purification of organelle DNA from a broad range of eukaryotes. By emphasizing the underlying principles, these procedures will facilitate the development of new species-specific protocols. The presented purification schemes involve either isolation of organelles and subsequent extraction of DNA from this subcellular fraction, or processing of whole-cell lysates followed by CsCl gradient centrifugation to separate nuclear and organelle DNAs according to their A + T content. We have successfully used the described procedures for organelle genome sequencing from diverse eukaryotes, including non-axenic protists. Procedures can be completed in 3–5 days, typically yielding a few micrograms of DNA—ample for sequencing complete genomes.

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Figure 1
Figure 2: Restriction analysis of DNA fractions obtained in a whole-cell lysate purification experiment with the protist Reclinomonas americana.

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References

  1. Anderson, S. et al. Sequence and organization of the human mitochondrial genome. Nature 290, 457–465 (1981).

    Article  CAS  Google Scholar 

  2. dePamphilis, C.W. & Palmer, J.D. Loss of photosynthetic and chlororespiratory genes from the plastid genome of a parasitic flowering plant. Nature 348, 337–339 (1990).

    Article  CAS  Google Scholar 

  3. O'Brien, E.A. et al. GOBASE—a database of organelle and bacterial genome information. Nucleic Acids Res. 34, D697–D699 (2006).

    Article  CAS  Google Scholar 

  4. Gray, M.W., Lang, B.F. & Burger, G. Mitochondria of protists. Annu. Rev. Genet. 38, 477–524 (2004).

    Article  CAS  Google Scholar 

  5. Boore, J.L. Animal mitochondrial genomes. Nucleic Acids Res. 27, 1767–1780 (1999).

    Article  CAS  Google Scholar 

  6. Burger, G., Saint-Louis, D., Gray, M.W. & Lang, B.F. Complete sequence of the mitochondrial DNA of the red alga Porphyra purpurea. Cyanobacterial introns and shared ancestry of red and green algae. Plant Cell 11, 1675–1694 (1999).

    Article  CAS  Google Scholar 

  7. Lang, B.F., O'Kelly, C., Nerad, T., Gray, M.W. & Burger, G. The closest unicellular relatives of animals. Curr. Biol. 12, 1773–1778 (2002).

    Article  CAS  Google Scholar 

  8. Martin, W. et al. Gene transfer to the nucleus and the evolution of chloroplasts. Nature 393, 162–165 (1998).

    Article  CAS  Google Scholar 

  9. Lin, X. et al. Sequence and analysis of chromosome 2 of the plant Arabidopsis thaliana . Nature 402, 761–768 (1999).

    Article  CAS  Google Scholar 

  10. Woischnik, M. & Moraes, C.T. Pattern of organization of human mitochondrial pseudogenes in the nuclear genome. Genome Res. 12, 885–893 (2002).

    Article  CAS  Google Scholar 

  11. Burger, G., Forget, L., Zhu, Y., Gray, M.W. & Lang, B.F. Unique mitochondrial genome architecture in unicellular relatives of animals. Proc. Natl. Acad. Sci. USA 100, 892–897 (2003).

    Article  CAS  Google Scholar 

  12. Marande, W., Lukes, J. & Burger, G. Unique mitochondrial genome structure in diplonemids, the sister group of kinetoplastids. Eukaryot. Cell 4, 1137–1146 (2005).

    Article  CAS  Google Scholar 

  13. Seif, E. et al. Comparative mitochondrial genomics in zygomycetes: bacteria-like RNase P RNAs, mobile elements and a close source of the group I intron invasion in angiosperms. Nucleic Acids Res. 33, 734–744 (2005).

    Article  CAS  Google Scholar 

  14. Kuck, U., Stahl, U., Lhermitte, A. & Esser, K. Isolation and characterization of mitochondrial DNA from the alkane yeast Saccharomycopsis lipolytica . Curr. Genet. 2, 97–101 (1980).

    Article  CAS  Google Scholar 

  15. Tabak, H.F. & Weijers, P.J. The genetic complexity of Schizosaccharomyces pombe mitochondrial DNA. FEBS Lett. 69, 211–215 (1976).

    Article  CAS  Google Scholar 

  16. Portugal, J. & Waring, M.J. Assignment of DNA binding sites for 4′,6-diamidine-2-phenylindole and Bisbenzimide (Hoechst 33258). A comparative footprinting study. Biochim. Biophys. Acta 949, 158–168 (1988).

    Article  CAS  Google Scholar 

  17. Garber, R.C. & Yoder, O.C. Isolation of DNA from filamentous fungi and separation into nuclear, mitochondrial, ribosomal, and plasmid components. Anal. Biochem. 135, 416–422 (1983).

    Article  CAS  Google Scholar 

  18. Hudspeth, M.E., Shumard, D.S., Tatti, K.M. & Grossman, L.I. Rapid purification of yeast mitochondrial DNA in high yield. Biochim. Biophys. Acta 610, 221–228 (1980).

    Article  CAS  Google Scholar 

  19. Turmel, M., Otis, C. & Lemieux, C. The complete chloroplast DNA sequence of the green alga Nephroselmis olivacea: insights into the architecture of ancestral chloroplast genomes. Proc. Natl. Acad. Sci. USA 96, 10248–10253 (1999).

    Article  CAS  Google Scholar 

  20. Hagopian, J.C., Nyvall, P. & de Oliveira, M.C. Purification of plastid DNA from an enriched fraction of the red alga Graciliaria tenuistipitata . Plant Mol. Biol. Reptr. 20, 399–406 (2002).

    Article  CAS  Google Scholar 

  21. Schramke, M.L. & Orlowski, M. Mitochondrial genome of the dimorphic zygomycete Mucor racemosus . Curr. Genet. 24, 337–343 (1993).

    Article  CAS  Google Scholar 

  22. Hackett, J.D. et al. Migration of the plastid genome to the nucleus in a peridinin dinoflagellate. Curr. Biol. 14, 213–218 (2004).

    Article  CAS  Google Scholar 

  23. Laatsch, T., Zauner, S., Stoebe-Maier, B., Kowallik, K.V. & Maier, U.G. Plastid-derived single gene minicircles of the dinoflagellate Ceratium horridum are localized in the nucleus. Mol. Biol. Evol. 21, 1318–1322 (2004).

    Article  CAS  Google Scholar 

  24. Wiesner, R.J., Swift, H. & Zak, R. Purification of mitochondrial DNA from total cellular DNA of small tissue samples. Gene 98, 277–281 (1991).

    Article  CAS  Google Scholar 

  25. Gilson, P., Waller, R. & McFadden, G. Preliminary characterisation of chlorarachniophyte mitochondrial DNA. J. Eukaryot. Microbiol. 42, 696–701 (1995).

    Article  CAS  Google Scholar 

  26. Backert, S., Dorfel, P. & Borner, T. Investigation of plant organellar DNAs by pulsed-field gel electrophoresis. Curr. Genet. 28, 390–399 (1995).

    Article  CAS  Google Scholar 

  27. Bendich, A.J. Reaching for the ring: the study of mitochondrial genome structure. Curr. Genet. 24, 279–290 (1993).

    Article  CAS  Google Scholar 

  28. Oldenburg, D.J. & Bendich, A.J. Mitochondrial DNA from the liverwort Marchantia polymorpha: circularly permuted linear molecules, head-to-tail concatamers, and a 5′ protein. J. Mol. Biol. 310, 549–562 (2001).

    Article  CAS  Google Scholar 

  29. Rodriguez-Ezpeleta, N., Teijeiro, S., Forget, L., Burger, G. & Lang, B.F. in Methods in Molecular Biology: Methods in ESTs (ed. Parkinson, J.) (Humana Press, Totowa, NJ, 2007).

    Google Scholar 

  30. Boore, J.L., Macey, J.R. & Medina, M. Sequencing and comparing whole mitochondrial genomes of animals. Methods Enzymol. 395, 311–348 (2005).

    Article  CAS  Google Scholar 

  31. Tapper, D.P., Van Etten, R.A. & Clayton, D.A. Isolation of mammalian mitochondrial DNA and RNA and cloning of the mitochondrial genome. Methods Enzymol. 97, 426–434 (1983).

    Article  CAS  Google Scholar 

  32. Simison, W.B., Lindberg, D.R. & Boore, J.L. Rolling circle amplification of metazoan mitochondrial genomes. Mol. Phylogenet. Evol. 39, 562–567 (2006).

    Article  CAS  Google Scholar 

  33. Palva, T.K. & Palva, E.T. Rapid isolation of animal mitochondrial DNA by alkaline extraction. FEBS Lett. 192, 267–270 (1985).

    Article  CAS  Google Scholar 

  34. DeSalle, R., Williams, A.K. & George, M. Isolation and characterization of animal mitochondrial DNA. Methods Enzymol. 224, 176–204 (1993).

    Article  CAS  Google Scholar 

  35. Peloquin, J.J., Bird, D.M. & Platzer, E.G. Rapid miniprep isolation of mitochondrial DNA from metacestodes, and free-living and parasitic nematodes. J. Parasitol. 79, 964–967 (1993).

    Article  CAS  Google Scholar 

  36. Burger, G., Lavrov, D.V., Forget, L. & Lang, B.F. Sequencing complete mitochondial and plastid genomes. Nat. Protoc. 2, 603–614 (2007).

    Article  CAS  Google Scholar 

  37. Scotti, N., Cardi, T. & Marechal-Drouard, L. Mitochondrial DNA and RNA isolation from small amounts of potato tissue. Plant Mol. Biol. Reptr. 19, 67a–67h (2001).

    Article  Google Scholar 

  38. Wilson, A.J. & Chourey, P.S. A rapid and inexpensive method for the isolation of restrictable mitochondrial DNA from various plant sources. Plant Cell Rep. 3, 237–239 (1984).

    Article  CAS  Google Scholar 

  39. Triboush, S.O., Danilenko, N.G. & Davydenko, O.G. A method for isolation of chloroplast DNA and mitochondrial DNA from sunflower. Plant Mol. Biol. Reptr. 16, 183–189 (1998).

    Article  CAS  Google Scholar 

  40. Kabeya, Y. & Sato, N. Unique translation initiation at the second AUG codon determines mitochondrial localization of the phage-type RNA polymerases in the moss Physcomitrella patens . Plant Physiol. 138, 369–382 (2005).

    Article  CAS  Google Scholar 

  41. McNeal, J.R. et al. Using partial genomic fosmid libraries for sequencing complete organellar genomes. Biotechniques 41, 69–73 (2006).

    Article  CAS  Google Scholar 

  42. Wang, W. et al. Construction of a bacterial artificial chromosome library from the spikemoss Selaginella moellendorffii: a new resource for plant comparative genomics. BMC Plant Biol. 5, 10 (2005).

    Article  Google Scholar 

  43. Bookjans, G., Stummann, B.M. & Henningsen, K.W. Preparation of chloroplast DNA from pea plastids isolated in a medium of high ionic strength. Anal. Biochem. 141, 244–247 (1984).

    Article  CAS  Google Scholar 

  44. Jansen, R.K. et al. Methods for obtaining and analyzing whole chloroplast genome sequences. Methods Enzymol. 395, 348–384 (2005).

    Article  CAS  Google Scholar 

  45. Mourad, G.S. Chloroplast DNA isolation. Methods Mol. Biol. 82, 71–77 (1998).

    CAS  PubMed  Google Scholar 

  46. van de Ven, M.T., Lanham, P.G. & Brennan, R.M. Isolation and purification of plant nucleic acids. Genomic and chloroplast DNA. Methods Mol. Biol. 50, 1–14 (1996).

    CAS  PubMed  Google Scholar 

  47. Fire, A. & Xu, S.Q. Rolling replication of short DNA circles. Proc. Natl. Acad. Sci. USA 92, 4641–4645 (1995).

    Article  CAS  Google Scholar 

  48. Querol, A. & Barrio, E. A rapid and simple method for the preparation of yeast mitochondrial DNA. Nucleic Acids Res. 18, 1657 (1990).

    Article  CAS  Google Scholar 

  49. Schafer, B., Hansen, M. & Lang, B.F. Transcription and RNA-processing in fission yeast mitochondria. RNA 11, 785–795 (2005).

    Article  Google Scholar 

  50. Lang, B. et al. A simple method for the large-scale preparation of mitochondria from microorganisms. Anal. Biochem. 77, 110–121 (1977).

    Article  CAS  Google Scholar 

  51. Cramer, C.L., Ristow, J.L., Paulus, T.J. & Davis, R.H. Methods for mycelial breakage and isolation of mitochondria and vacuoles of Neurospora . Anal. Biochem. 128, 384–392 (1983).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Dr. M. Reith (Halifax) for providing us with his variant of CTAB DNA purification method that he optimized for red algae. This work was supported by the Canadian Institute of Health Research (B.F.L., grant MOP-42475; G.B., grant MOP-79309). Interaction support from the Canadian Institute of Advanced Research, Program in Evolutionary Biology (G.B. and B.F.L.) is gratefully acknowledged.

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  1. Département de Biochimie, Robert Cedergren Center for Bioinformatics and Genomics, Canadian Institute for Advanced Research, Program in Evolutionary Biology, Université de Montréal 2900 Boulevard Edouard-Montpetit, CP 6128, Montréal, H3T 1J4, Québec, Canada

    B Franz Lang & Gertraud Burger

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Correspondence to B Franz Lang.

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Lang, B., Burger, G. Purification of mitochondrial and plastid DNA. Nat Protoc 2, 652–660 (2007). https://doi.org/10.1038/nprot.2007.58

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