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Ancient DNA in Sediments

Ancient DNA in Sediments. Department of Evolutionary Biology Zoological Institute University of Copenhagen. Ancient DNA Studies. DNA from Sediments. Sample Information. Microscopy. Cells in the bacterial size range (about 10 7 cells/ gww, average cell volume 0.03-0.05 µm 3 /cell)

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Ancient DNA in Sediments

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  1. Ancient DNA in Sediments Department of Evolutionary Biology Zoological Institute University of Copenhagen

  2. Ancient DNA Studies

  3. DNA from Sediments

  4. Sample Information

  5. Microscopy • Cells in the bacterial size range (about 107cells/ gww, average cell volume 0.03-0.05 µm3/cell) • Occasional fine rootlets (≥2 mm in diameter), seeds and small unidentifiable multicellular fragments • No bone/hair/identifiable animal soft tissue

  6. PCR Based Analyses • 4 x 0.25gww soil • FAST PREP • DNA extraction/purification • PCR (“universal”/”specific” primers for rbcL/mtDNA) • Cloning • Sequencing • BLAST (GenBank)/phylogenetic analysis

  7. Precaution, Controls, Criteria • Special rotation-column coring method • Spiking with bacterial Serratia marcescens • Isolated, dedicated clean lab. • Isolated ventilation system, UV-radiation, flow hood • Facemasks, gamma-sterilized glows, hats • Removal of core surfaces • Cleaning of reagents/tools: UV, HCL, bleach, ultrafiltration • Extraction/ PCR controls • Cloning • Independent reproducibility of results • Phylogenetic criteria

  8. Important! Not previously worked with in the Copenhagen lab (at that stage): • plant rbcL DNA • DNA from Arctic or NZ animals (including megafauna) except for Reindeer mtDNA Previously produced PCR products is a major source of contamination

  9. Amplification Results Plants (rbcL about 130 bp): • PCR products up to 300-400 kyr (including NZ cave site) • No PCR products million year old samples Animal (mtDNA 88-234 bp): • PCR products up to 20-30 kyr (including NZ cave site, only primers for bird mtDNA) • no PCR products 300-400 kyr and million year old samples The results were independently confirmed in Oxford

  10. Plant identifications(multiple GenBank sequences showing >96% similarity to the clones; reproducibility confirmed by a bootstrap test )

  11. Source of rbcL DNA • Chloroplast sequences are essentially absent from angiosperm pollen (Blanchard & Schmidt 1995) • The majority of the plant sequences must originate from locally deposited seeds, or somatic tissue such as the observed fine rootlets

  12. mtDNA 16S (88-95 bp)

  13. Control mtDNA region (124-129bp)

  14. mtDNA cyt b sequences (A, 98 bp and B, 229 bp)

  15. Control mtDNA region (202-203 bp)

  16. Control mtDNA region 234 bp

  17. Source of Animal mtDNA Unknown Dung is a possibility? From Poinar et al. (2001)

  18. Plant Sequence Diversity(>96% similarity)

  19. Frequency; Herbs, Shrubs, Mosses

  20. Conclusions • Diverse ancient DNA directly from soil (even in the absence of obvious microfossils) • Change in plant diversity (following climate change) • Change in herb/shrub dominance • Change in Poaceae and Cyperaceae frequency (Pleistocene/Holocene boundary) • Megafauna present during LGM • DNA better preserved in permafrost than cave sediments • Clutha River vegetation cover similar to pre-human occupation of NZ even at 600 kyr

  21. Combined with pollen records and fossil bones revealing Paleobiological change Genetic information from archaeological records even in the absence of macrofossil evidence? Perspectives

  22. DNA damage analysis • DNA in fossil remains is known to be degraded • Unknown to a large extent what types of damages accumulate • And especially what types of damages prevents amplification of DNA

  23. DNA breaks

  24. Interstrand Crosslinks(Denaturation experiment)

  25. Rate constants

  26. Conclusion • DNA in permanently frozen sediments are degraded by alkylation and hydrolysis, producing single and double stranded breaks as well as interstrand crosslinks • ICL accumulate more rapidly than SSB • SSB is generated by depurination • The observed damage pattern indicate that DNA degradation result from spontaneous rather than exogenous processes.

  27. Perspectives Repair of ancient DNA Possible dating of sampels Determination of spontaneous accumulation of DNA damages in cells

  28. Alan Cooper Anders J. Hansen Beth Shapiro Carsten Wiuf David A. Gilichinsky David Mitchell Eske Willerslev Jonas Binladen Lakshmi Paniker M. Thomas P. Gilbert Mike Bunce Regin Rønn Tina B. Brand Department of Evolutionary Biology, Zoological Institute, University of Copenhagen, Denmark Henry Wellcome Ancient Biomolecules Centre, Department of Zoology, University of Oxford, UK Department of Statistics, University of Oxford, UK Soil Cryology Laboratory, Institute for PhysicoChemical and Biological Problems in Soil Science, Russian Academy of Sciences, Russsia Department of Cariogenese, MDAnderson Cancer institute, UT The work has been done by:

  29. Beringia

  30. Beringia Megafauna of the Late Pleistocene

  31. Arctic Dessert or Steppe?Why Megafauna got Extinct?

  32. Traditional Approach Pollen analyses Problems: Variation in influx rates, long distance dispersal, no account for vegetative growth, problems of taxonomic identification Vertebrate fossils Problems: Different preservation, dating beyond carbon age

  33. Thoughts… • Is it possible to address the paleo- environment of Beringia by obtaining DNA directly from the permafrost sediments even in the absence of macrofossils? • Cold conditions is critical for the long-term preservation of DNA (Smith et al. 2002). If plant or animal DNA accumulates in sediments permafrost must provide ideal preservation conditions

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