Biochemical Genetics

, Volume 35, Issue 5–6, pp 165–179 | Cite as

Proboscidean DNA from Museum and Fossil Specimens: An Assessment of Ancient DNA Extraction and Amplification Techniques

  • Hong Yang
  • Edward M. Golenberg
  • Jeheskel Shoshani


Applications of reliable DNA extraction and amplification techniques to postmortem samples are critical to ancient DNA research. Commonly used methods for isolating DNA from ancient material were tested and compared using both soft tissue and bones from fossil and contemporary museum proboscideans. DNAs isolated using three principal methods served as templates in subsequent PCR amplifications, and the PCR products were directly sequenced. Authentication of the ancient origin of obtained nucleotide sequences was established by demonstrating reproducibility under a blind testing system and by phylogenetic analysis. Our results indicate that ancient samples may respond differently to extraction buffers or purification procedures, and no single method was universally successful. A CTAB buffer method, modified from plant DNA extraction protocols, was found to have the highest success rate. Nested PCR was shown to be a reliable approach to amplify ancient DNA templates that failed in primary amplification.

ancient DNA cetyltrimethylammonium bromide extraction nested polymerase chain reaction proboscideans 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Barnard, G. F., Puder, M., Begum, N. A., and Chen, L. B. (1994). PCR product sequencing with [α-33P] and [α-32P] dATP. BioTechniques 16:572.Google Scholar
  2. Cano, R. J., and Poinar, H. N. (1993). Rapid isolation of DNA from fossil and museum specimens suitable for PCR. BioTechniques 15:432.Google Scholar
  3. Cattano, C., Smillie, D. M., Gelsthorpe, K., Piccinini, A., Gelsthorpe, A. R., and Sokol, R. J. (1995). A simple method for extracting DNA from old skeletal material. Forens. Sci. Instr. 74:167.Google Scholar
  4. Cooper, A. (1994). DNA from museum specimens. In Herrmann, B., and Hummel, S. (eds.), Ancient DNA, Springer-Verlag, New York, pp. 149–165.Google Scholar
  5. Cooper, A., Mourer-Chauvire, C., Chambers, G. K., von Haeseler, A., Wilson, A. C., and Pääbo, S. (1992). Independent origins of New Zealand moas and kiwis. Proc. Natl. Acad. Sci. USA 89:8741.Google Scholar
  6. Doran, G. H., Dickel, D. N., Ballinger, W. E., Jr., Agee, O. F., Laipis, P. J., and Hauswirth, W. W. (1986). 8000 year old human brain tissue: anatomical cellular and molecular analysis. Nature 323:803.Google Scholar
  7. Doyle, J. J., and Doyle, J. L. (1987). A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19:11.Google Scholar
  8. Felsenstein, J. (1990). PHYLIP (Phylogeny Inference Package), Version 3.3, Computer program distributed by University of Washington, Seattle, WA.Google Scholar
  9. Gaensslen, R. E., and Berka, K. (1994). Studies on DNA polymorphisms in human bone and soft tissues. Anal. Chem. Acta 288:3.Google Scholar
  10. Golenberg, E. M. (1994). Fossil samples: DNA from plant compression fossils. In Herrmann, B., and Hummel, S. (eds.), Ancient DNA, Springer Verlag, New York, pp. 233–252.Google Scholar
  11. Golenberg, E. M., Bickel, A., and Wiehs, P. (1996). Effect of highly fragmented DNA on PCR. Nucleic Acids Res. 24:5026.Google Scholar
  12. Hagelberg, E. (1994). Mitochondrial DNA from Ancient bones. In Herrmann, B., and Hummel, S. (eds.), Ancient DNA, Springer-Verlag, New York, pp. 195–204.Google Scholar
  13. Handt, O., Richards, M., Trommsdorff, M., Kilger, C., Simanainen, J., Georgiev, O., Bauer, K., Stone, A., Hedges, R., Schaffner, W., Utermann, G., Sykes, B., and Pääbo, S. (1994). Molecular genetic analyses of the Tyrolean Ice Man. Science 264:1775.Google Scholar
  14. Hänni, C., Laudet, V., Stehelin, D., and Taberlet, P. (1994). Tracking the origins of the cave bear (Ursus spelaeus) by mitochondrial DNA sequencing. Proc. Natl. Acad. Sci. USA 91:12336.Google Scholar
  15. Herrmann, B., and Hummel, S. (eds.) (1994). Ancient DNA, Springer-Verlag, New York.Google Scholar
  16. Höss, M., and Pääbo, S. (1993). DNA extraction from Pleistocene bones by a silica-based purification method. Nucleic Acids Res. 21:3913.Google Scholar
  17. Höss, M., Dilling, A., Currant, A., and Pääbo, S. (1996). Molecular phylogeny of the extinct ground sloth Mylodon darwinii. Proc. Natl. Acad. Sci. USA 93:181.Google Scholar
  18. Hummel, S., Nordsiek, G., and Herrmann, B. (1992). Improved efficiency in amplification of ancient DNA and its sequence analysis. Naturwissenschaften 79:359.Google Scholar
  19. Irwin, D. M., Kocher, T. D., and Wilson, A. C. (1991). Evolution of the cytochrome b gene of mammals. J. Mol. Evol. 32:128.Google Scholar
  20. Janczewski, D. N., Yuhki, N., Gilbert, D. A., Jefferson, G. T., and O'Brien, S. J. (1992). Molecular phylogenetic inference from saber-toothed cat fossils of Rancho La Brea. Proc. Natl. Acad. Sci. USA 89:9769.Google Scholar
  21. Krajewski, C., Driskell, A. C., Baverstock, P. R., and Braun, M. J. (1992). Phylogenetic relationships of the thylacine (Mammalia:Thylacinidae) among dasyuroid marsupials: Evidence from cytochrome b DNA sequences. Proc. R. Soc. Lond. B 250:19.Google Scholar
  22. Lowenstein, J. M., and Shoshani, J. (1996). Proboscidean relationships based on immunological data. In Shoshani, J., and Tassy, P. (eds.), The Proboscidea: Evolution and Palaeoecology of Elephants and Their Relatives, Oxford University Press, Oxford, pp. 49–54.Google Scholar
  23. Pääbo, S. (1989). Ancient DNA: Extraction, characterization, molecular cloning, and enzymatic amplification. Proc. Natl. Acad. Sci. USA 86:1939.Google Scholar
  24. Pääbo, S., Gifford, J. A., and Wilson, A. C. (1988). Mitochondrial DNA sequences from a 7000-year old brain. Nucleic Acids Res. 16:9775.Google Scholar
  25. Pääbo, S., Higuchi, R. G., and Wilson, A. C. (1989). Ancient DNA and the polymerase chain reaction. J. Biol. Chem. 264:9709.Google Scholar
  26. Rogers, S. O. (1994). Phylogenetic and taxonomic information from herbarium and mummified DNA. In Adams, R. P., Miller, J. S., Golenberg, E. M., and Adams, J. E. (eds.), Conservation of Plant Genes II: Utilization of Ancient and Modern DNA, Missouri Botanical Garden, pp. 47–67.Google Scholar
  27. Salo, W. L., Aufderheide, A. C., Buikstra, J., and Holcomb, T. (1994). Identification of Mycobacterium tuberculosis DNA in a pre-Columbian Peruvian mummy. Proc. Natl. Acad. Sci. USA 91:2091.Google Scholar
  28. Shoshani, J. (1996). Para-or monophyly of the gomphotheres and their position within Proboscidea. In Shoshani, J., and Tassy, P. (eds.), The Proboscidea: Evolution and Palaeoecology of Elephants and Their Relatives, Oxford University Press, Oxford, pp. 149–177.Google Scholar
  29. Shoshani, J., et al. (1982). On the dissection of a female Asian elephant (Elephas maximus maximus Linnaeus, 1758) and data from other elephants. Elephant 2:3.Google Scholar
  30. Shoshani, J., Lowenstein, J. M., Walz, D. A., and Goodman, M. (1985). Proboscidean origins of mastodon and woolly mammoth demonstrated immunologically. Paleobiology 11:429.Google Scholar
  31. Shoshani, J., Fisher, D. C., Zawiskie, J. M., Thurlow, S. J., Shoshani, S. L., Benninghoff, W. S., and Zoch, F. H. (1989). The Shelton Mastodon Site: Multidisciplinary study of a late Pleistocene (Twocreekan) locality in southeastern Michigan. Contrib. Mus. Paleontol. Univ. Mich. 27:393.Google Scholar
  32. Swofford, D. L. (1991). PAUP: Phylogenetic Analysis Using Parsimony, Version 3.0, Computer program distributed by the Illinois Natural History Survey, Champaign, IL.Google Scholar
  33. Tassy, P. (1996). Who is who among the Proboscidea. In Shoshani, J., and Tassy, P. (eds.), The Proboscidea: Evolution and Palaeoecology of Elephants and Their Relatives, Oxford University Press, Oxford. pp. 39–48.Google Scholar
  34. Thomas, R. H. (1994). Molecules, museums and vouchers. Trends Ecol. Evol. 9:413.Google Scholar
  35. Tuross, N. (1994). The biochemistry of ancient DNA in bone. Experientia 50:530.Google Scholar
  36. Vochot, A.-M., and Monnerot, M. (1996). Extraction, amplification and sequencing of DNA from formaldehyde-fixed specimens. Ancient Biomolecules 1:3.Google Scholar
  37. Wayne, R. K., and Jenks, S. M. (1991). Mitochondrial DNA analysis implying extensive hybridization of the endangered red wolf Canis rufus. Nature 351:565.Google Scholar
  38. Yang, H., Golenberg, E. M., and Shoshani, J. (1996). Phylogenetic resolution within Elephantidae using fossil DNA sequence from American mastodon (Mammut americanum) as an outgroup. Proc. Natl. Acad. Sci. USA 93:1190.Google Scholar
  39. Yang, H., Golenberg, E. M., and Shoshani, J. (1997). A blind testing design for authenticating ancient DNA sequences. Mol. Phylogenies Evol. (in press).Google Scholar
  40. Zimmermann, K., Pischinger, K., and Mannhalter, J. W. (1994). Nested primer PCR detection limits of HIV-1 in the background of increasing numbers of lysed cells. BioTechniques 17:18.Google Scholar

Copyright information

© Plenum Publishing Corporation 1997

Authors and Affiliations

  • Hong Yang
    • 1
  • Edward M. Golenberg
    • 1
  • Jeheskel Shoshani
    • 1
    • 2
  1. 1.Department of Biological SciencesWayne State UniversityDetroit
  2. 2.Cranbrook Institute of ScienceBloomfield Hills

Personalised recommendations