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DNA Recovery from Formalin-Fixed Specimens

DNA Recovery from Formalin-Fixed Specimens. Sponsored by the Consortium for the Barcode of Life. DNA Barcode: short standardized sequence enabling species discrimination. DNA Barcode: short standardized sequence enabling species discrimination.

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DNA Recovery from Formalin-Fixed Specimens

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  1. DNA Recovery from Formalin-Fixed Specimens Sponsored by the Consortium for the Barcode of Life

  2. DNA Barcode:short standardized sequence enabling species discrimination

  3. DNA Barcode:short standardized sequence enabling species discrimination

  4. Barcodes: Developing a ReferenceLibrary for Known Species • Master key • ID all life stages • IDs cheap & fast • Residual taxonomic uncertainty low

  5. DNA Recovery from Formalin-Fixed Specimens

  6. Formalin Fixation • Routinely used in museum curation and pathology since ~1900 • 100s of millions of dried, paraffin-embedded tissue samples • Museum collections of fish, marine invertebrates, others • Principal obstacle to FISH-BOL, other barcoding projects, Census of Marine Life, biomedical research, AToL, etc.

  7. NRC Workshop • Organized by CBOL, co-funded by: • USDA and EPA • MCZ, Harvard and NESCENT, Duke Univ. • New England Biolabs and Sigma-Aldrich • Workshop Committee: • Ann Bucklin (UConn biol. oceanographer), co-chair • Don Crothers (Yale chemist), co-chair • Chris Schander (Univ. Bergen, Norway) • Tim O’Leary (Veterans Admin pathologist) • Alison Williams (Princeton biochemist) • 25 Participants: Curators, taxonomists, chemists

  8. Workshop Agenda • Review past efforts to recover DNA • Brainstorm on possible obstacles • Develop research agenda aimed at: • Understanding degradation processes • Improving extraction protocols • Developing repair enzymes, reversal processes • Exploring the use of new technologies • Engaging bioinformatics to reassemble fragments

  9. Major findings (1) • A variety of degradation processes and chemical obstacles are at work: • Cross-linking with proteins • Oxidation • Acidification • Depurination • Cytosine deamination • Formalin-ethanol interaction • Presence of PCR inhibitors • Point mutations • Denaturation

  10. Major findings (2) • Different degradation processes may leave chemical/physical signatures • Some degradation processes may be reversible, or damage may be repairable • Curatorial practices vary widely • Relation between curation and degradation processes can be established • Some specimens will be hopeless (pH 2.0)

  11. Major findings (3) • Taxonomists have created “cottage hobby” to extract DNA from formalinized tissue • More systematic experimental approach is needed • Chemical/physical indicators could: • Identify most promising specimens • Suggest optimal extraction procedures • Lead to improved fixation methods • Identify hopeless specimens

  12. Next Steps (1) • Understand curatorial processes: How has formalin been used (and is being used) in museums? • Survey selected museums • Understand degradation processes: Which processes are at work, and what indicators do they leave? • Analysis of Smithsonian “goldfish time capsule” • Analysis of museum specimens with well-documented curatorial histories

  13. Next Steps (2) • Characterize formalinized specimens: How does their chemistry vary? • Develop battery of chemical/physical indicators linked to degradation processes • pH, NMR, fragment size, free purines, others • Develop a public knowledge base of extraction protocols: What works and what doesn’t? • Compile published studies • Call for information on unpublished studies • Link curatorial history and extraction method to success or failure of DNA recovery

  14. Next Steps (3) • Test extraction methods in context: Which methods work relative to curatorial history and indicator data? • Create network of cooperating labs, museums • Standardize battery of extraction protocols • Standardize collection of curatorial histories and indicator data (like patient history and vital signs) • Calibrate labs using “Goldfish Standard” • Test across extraction methods, curatorial histories, indicator data

  15. Interested in Participating? • CBOL and SPNHC will collaborate • Contact CBOL Secretariat Office. Write to: • CBOLFormalin@si.edu or • David Schindel, CBOL Executive SecretarySchindelD@si.edu • Andrew Bentley, Collection Manager, Ichthyology, KU Natural History Museum ABentley@ku.edu

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