Transcript DNA Recovery from Formalin

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
Barcodes: Developing a Reference
Library for Known Species
•Master key
•ID all life stages
•IDs cheap & fast
•Residual taxonomic uncertainty low
DNA Recovery from
Formalin-Fixed Specimens
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.
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:
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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
Workshop Agenda
• Review past efforts to recover DNA
• Brainstorm on possible obstacles
• Develop research agenda aimed at:
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Understanding degradation processes
Improving extraction protocols
Developing repair enzymes, reversal processes
Exploring the use of new technologies
Engaging bioinformatics to reassemble fragments
Major findings (1)
• A variety of degradation processes and chemical
obstacles are at work:
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Cross-linking with proteins
Oxidation
Acidification
Depurination
Cytosine deamination
Formalin-ethanol interaction
Presence of PCR inhibitors
Point mutations
Denaturation
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)
Major findings (3)
• Taxonomists have created “cottage hobby” to
extract DNA from formalinized tissue
• More systematic experimental approach is needed
• Chemical/physical indicators could:
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Identify most promising specimens
Suggest optimal extraction procedures
Lead to improved fixation methods
Identify hopeless specimens
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
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
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
Interested in Participating?
• CBOL and SPNHC will collaborate
• Contact CBOL Secretariat Office.
Write to:
– [email protected] or
– David Schindel, CBOL Executive Secretary
[email protected]
– Andrew Bentley, Collection Manager, Ichthyology,
KU Natural History Museum
[email protected]