DNA Recovery from Formalin-Fixed Specimens

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DNA Recovery from Formalin-Fixed Specimens

• Sponsored by the Consortium for the Barcode of
  Life

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DNA Barcodeshort standardized sequence enabling
species discrimination
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DNA Barcodeshort standardized sequence enabling
species discrimination
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Barcodes Developing a ReferenceLibrary for
Known Species

• Master key
• ID all life stages
• IDs cheap fast
• Residual taxonomic uncertainty low

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DNA Recovery from Formalin-Fixed Specimens
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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.

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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 OLeary (Veterans Admin pathologist)
• Alison Williams (Princeton biochemist)
• 25 Participants Curators, taxonomists, chemists

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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

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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

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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)

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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

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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

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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 doesnt?
• Compile published studies
• Call for information on unpublished studies
• Link curatorial history and extraction method to
  success or failure of DNA recovery

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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

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Interested in Participating?

• CBOL and SPNHC will collaborate
• Contact CBOL Secretariat Office. Write to
• CBOLFormalin_at_si.edu or
• David Schindel, CBOL Executive SecretarySchindelD
  _at_si.edu
• Andrew Bentley, Collection Manager, Ichthyology,
• KU Natural History Museum
• ABentley_at_ku.edu