PATH TO EFFECTIVE RECOVERING OF DNA FROM FORMALIN-FIXED BIOLOGICAL SAMPLES IN NATURAL HISTORY COLLECTIONS
WORKSHOP SUMMARY
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NOTICE: The project that is the subject of this report was approved by the Governing Board of the National Research Council, whose members are drawn from the councils of the National Academy of Sciences, the National Academy of Engineering, and the Institute of Medicine. The members of the committee responsible for the report were chosen for their special competences and with regard for appropriate balance.
This study was supported by the Consortium for the Barcode of Life, the Museum of Comparative Zoology of Harvard University, the National Evolutionary Synthesis Center, New England Biolabs, Inc., Sigma-Aldrich Company, the U.S. Department of Agriculture’s Agricultural Research Service, and the U.S. Environmental Protection Agency’s Environmental Monitoring and Assessment Program. The content of this publication does not necessarily reflect the views or policies of the organizations or agencies that provide support for the project, nor does mention of trade names, commercial products or organizations imply endorsement by the agencies or organizations.
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Cover: Cover design by Michele de la Menardiere. Photo of fish collection by Chip Clark, the National Museum of Natural History, the Smithsonian Institution.
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THE NATIONAL ACADEMIES
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STEERING COMMITTEE FOR THE WORKSHOP ON RECOVERING DNA FROM FORMALIN-FIXED BIOLOGICAL SAMPLES
ANN C. BUCKLIN (Cochair),
University of Connecticut, Groton
DONALD M. CROTHERS (Cochair),
Yale University, New Haven, Connecticut
TIMOTHY O’LEARY,
U.S. Department of Veterans Affairs, Washington, D.C.
CHRISTOFFER SCHANDER,
University of Bergen, Norway
ALISON WILLIAMS,
Princeton University, New Jersey
Staff
EVONNE P.Y. TANG, Study Director
FRANCES E. SHARPLES, Director, Board on Life Sciences
TOVA G. JACOBOVITS, Senior Program Assistant
ANNE F. JURKOWSKI, Senior Program Assistant
KATE KELLY, Editor
BOARD ON LIFE SCIENCES
KEITH YAMAMOTO (Chair),
University of California, San Francisco
ANN M. ARVIN,
Stanford University School of Medicine, Stanford, California
JEFFREY L. BENNETZEN,
University of Georgia, Athens
RUTH BERKELMAN,
Emory University, Atlanta, Georgia
DEBORAH BLUM,
University of Wisconsin, Madison
R. ALTA CHARO,
University of Wisconsin, Madison
JEFFREY L. DANGL,
University of North Carolina, Chapel Hill
PAUL R. EHRLICH,
Stanford University, Stanford, California
MARK D. FITZSIMMONS,
John D. and Catherine T. MacArthur Foundation, Chicago, Illinois
JO HANDELSMAN,
University of Wisconsin, Madison
ED HARLOW,
Harvard Medical School, Boston, Massachusetts
KENNETH H. KELLER,
University of Minnesota, Minneapolis
RANDALL MURCH,
Virginia Polytechnic Institute and State University, Alexandria
GREGORY A. PETSKO,
Brandeis University, Waltham, Massachusetts
MURIEL E. POSTON,
Skidmore College, Saratoga Springs, New York
JAMES REICHMAN,
University of California, Santa Barbara
MARC T. TESSIER-LAVIGNE,
Genentech, Inc., South San Francisco, California
JAMES TIEDJE,
Michigan State University, East Lansing
TERRY L. YATES,
University of New Mexico, Albuquerque
Staff
FRANCES E. SHARPLES, Director
KERRY A. BRENNER, Senior Program Officer
ADAM P. FAGEN, Program Officer
ANNA FARRAR, Financial Associate
TOVA G. JACOBOVITS, Senior Program Assistant
ANNE F. JURKOWSKI, Senior Program Assistant
ANN H. REID, Senior Program Officer
MARILEE K. SHELTON-DAVENPORT, Senior Program Officer
EVONNE P.Y. TANG, Senior Program Officer
ROBERT T. YUAN, Senior Program Officer
Preface
Museums catalogue our knowledge of the Earth’s biodiversity, and their collections represent many decades of work by experts. Access to DNA sequence information in archival specimens would greatly extend knowledge of the genetic relationships within our biosphere. However, molecular genetic analysis of museum specimens has been slowed by the usual practice of fixation of samples in formalin and storage in alcohol or formalin. The fixation and storage induce changes in DNA that are not fully understood. With more frequent use of morphological and molecular characters for taxonomic and systematic analysis, the “formalin problem” has grown in significance. For example, a global effort to determine DNA barcodes (short DNA sequences for species recognition and discovery) for life on earth could be markedly expedited by sequencing DNA from specimens in museum collections. Molecular analysis of formalin-fixed tissue would allow biologists to address retrospective questions about how patterns in the genetic diversity of plant and animal species have changed over time. With application of molecular genetic analysis to formalin-fixed museum specimens, new insights about biodiversity, population dynamics, and ecosystem function can be gained from collections sampled years and perhaps decades ago.
There are many technical challenges to solving the “formalin problem,” beginning with the wide variation in curatorial practices for specimen storage. Some organisms are fixed in formalin only for a short time and then transferred to alcohol for long-term storage; others are fixed and stored in
formalin permanently. The rapid reactions of formalin with double helical DNA generally are reversible, but over the long term, especially with denaturation of the DNA, a variety of reactions can occur, many of which have not been characterized. Those reactions can be irreversible, and they can either mask the nature of the modified nucleotide in enzymatic replication, or they can block chain elongation altogether, resulting in failure of polymerase chain reaction amplification. To further complicate matters, oxidation of formaldehyde in formalin to formic acid produces an acidic solution in which depurination reactions and subsequent chain scission are greatly accelerated. Given the variation in preservation practices, and the variable age of the samples, it is unlikely that the “formalin problem” can be solved for all samples. However, with better understanding of the chemistry of formalin reactions and their effect on DNA integrity, and with better knowledge of curatorial history and practices, it should be possible to select likely candidates for intensive DNA isolation and sequencing experiments, with the goal of reconstructing significant portions of the genome.
On May 8-9, 2006, the Board on Life Sciences of the National Academies convened a workshop, “Recovering DNA from Formalin-Fixed Biological Samples.” Participants were experts—biophysicists, chemists, molecular biologists, and bioinformaticians—who discussed the path to successfully obtaining DNA sequence information from formalin-fixed biological samples. Unlike study committees of the National Research Council, workshops do not reach conclusions or present recommendations. However, participants in this workshop spent much time considering the research and experimentation that could be done to advance retrieval of genomic information from formalin-fixed samples. We thank all of the workshop participants for sharing their expertise and experience and for the stimulating discussions and insightful suggestions.
Ann C. Bucklin
Donald M. Crothers
Cochairs, Steering Committee for the
Workshop on Recovering DNA from
Formalin-Fixed Biological Samples
Acknowledgments
This document presents the author’s summary of the workshop discussion and does not necessarily reflect the views of the roundtable members or other participants.
This summary has been reviewed in draft form by individuals chosen for their diverse perspectives and technical expertise, in accordance with procedures approved by the National Research Council Report Review Committee. This independent review is intended to provide candid and critical comments that will assist the institution in making its published workshop summary as sound as possible and to ensure that the workshop summary meets institutional standards for objectivity, evidence, and responsiveness to the study charge. The review comments and draft manuscript remain confidential to protect the integrity of the deliberative process. We wish to thank the following individuals for their review of this workshop summary:
Robert DeSalle, American Museum of Natural History
Neil Hall, The Institute for Genomic Research
Jack Lichy, The Veterans Affairs Medical Center
Stephen Quake, Stanford University
Gary Rosenberg, Academy of Natural Sciences
Although the reviewers have provided many constructive comments and suggestions, they were not asked to endorse the content nor did they
see the final draft of the workshop summary before its release. The review of this workshop summary was overseen by Marvalee Wake, University of California, Berkeley. Appointed by the National Research Council, she was responsible for making certain that an independent examination of this workshop summary was carried out in accordance with institutional procedures and that all review comments were carefully considered. Responsibility for the final content of this workshop summary rests entirely with the institution.