DNA Recovery from Formalin-Fixed Specimens

Sponsored by the by the

Consortium for the Barcode of Consortium for the Barcode of

LifeLife

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:– 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:

– 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:– 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:– 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:– CBOLFormalin@si.edu or– David Schindel, CBOL Executive Secretary

SchindelD@si.edu– Andrew Bentley, Collection Manager,

Ichthyology,

KU Natural History Museum ABentley@ku.edu