PATH TO EFFECTIVE RECOVERING OF DNA FROM FORMALIN-FIXED BIOLOGICAL SAMPLES IN NATURAL HISTORY COLLECTIONS

WORKSHOP SUMMARY

Evonne P.Y. Tang

Board on Life Sciences

Division on Earth and Life Studies

NATIONAL RESEARCH COUNCIL OF THE NATIONAL ACADEMIES

THE NATIONAL ACADEMIES PRESS

Washington, D.C.
www.nap.edu



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Path to Effective Recovering of DNA from Formalin-Fixed Biological Samples in Natural History Collection: Workshop Summary PATH TO EFFECTIVE RECOVERING OF DNA FROM FORMALIN-FIXED BIOLOGICAL SAMPLES IN NATURAL HISTORY COLLECTIONS WORKSHOP SUMMARY Evonne P.Y. Tang Board on Life Sciences Division on Earth and Life Studies NATIONAL RESEARCH COUNCIL OF THE NATIONAL ACADEMIES THE NATIONAL ACADEMIES PRESS Washington, D.C. www.nap.edu

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Path to Effective Recovering of DNA from Formalin-Fixed Biological Samples in Natural History Collection: Workshop Summary THE NATIONAL ACADEMIES PRESS 500 Fifth Street, N.W. Washington, DC 20001 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. International Standard Book Number 0-309-10293-6 Cover: Cover design by Michele de la Menardiere. Photo of fish collection by Chip Clark, the National Museum of Natural History, the Smithsonian Institution. Additional copies of this report are available from the National Academies Press, 500 Fifth Street, N.W., Lockbox 285, Washington, DC 20055; (800) 624-6242 or (202) 334-3313 (in the Washington metropolitan area); Internet, http://www.nap.edu. Copyright 2006 by the National Academy of Sciences. All rights reserved.

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Path to Effective Recovering of DNA from Formalin-Fixed Biological Samples in Natural History Collection: Workshop Summary THE NATIONAL ACADEMIES Advisers to the Nation on Science, Engineering, and Medicine The National Academy of Sciences is a private, nonprofit, self-perpetuating society of distinguished scholars engaged in scientific and engineering research, dedicated to the furtherance of science and technology and to their use for the general welfare. Upon the authority of the charter granted to it by the Congress in 1863, the Academy has a mandate that requires it to advise the federal government on scientific and technical matters. Dr. Ralph J. Cicerone is president of the National Academy of Sciences. The National Academy of Engineering was established in 1964, under the charter of the National Academy of Sciences, as a parallel organization of outstanding engineers. It is autonomous in its administration and in the selection of its members, sharing with the National Academy of Sciences the responsibility for advising the federal government. The National Academy of Engineering also sponsors engineering programs aimed at meeting national needs, encourages education and research, and recognizes the superior achievements of engineers. Dr. Wm. A. Wulf is president of the National Academy of Engineering. The Institute of Medicine was established in 1970 by the National Academy of Sciences to secure the services of eminent members of appropriate professions in the examination of policy matters pertaining to the health of the public. The Institute acts under the responsibility given to the National Academy of Sciences by its congressional charter to be an adviser to the federal government and, upon its own initiative, to identify issues of medical care, research, and education. Dr. Harvey V. Fineberg is president of the Institute of Medicine. The National Research Council was organized by the National Academy of Sciences in 1916 to associate the broad community of science and technology with the Academy’s purposes of furthering knowledge and advising the federal government. Functioning in accordance with general policies determined by the Academy, the Council has become the principal operating agency of both the National Academy of Sciences and the National Academy of Engineering in providing services to the government, the public, and the scientific and engineering communities. The Council is administered jointly by both Academies and the Institute of Medicine. Dr. Ralph J. Cicerone and Dr. Wm. A. Wulf are chair and vice chair, respectively, of the National Research Council. www.national-academies.org

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Path to Effective Recovering of DNA from Formalin-Fixed Biological Samples in Natural History Collection: Workshop Summary 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

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Path to Effective Recovering of DNA from Formalin-Fixed Biological Samples in Natural History Collection: Workshop Summary 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

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Path to Effective Recovering of DNA from Formalin-Fixed Biological Samples in Natural History Collection: Workshop Summary 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

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Path to Effective Recovering of DNA from Formalin-Fixed Biological Samples in Natural History Collection: Workshop Summary 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

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Path to Effective Recovering of DNA from Formalin-Fixed Biological Samples in Natural History Collection: Workshop Summary 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

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Path to Effective Recovering of DNA from Formalin-Fixed Biological Samples in Natural History Collection: Workshop Summary 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.

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Path to Effective Recovering of DNA from Formalin-Fixed Biological Samples in Natural History Collection: Workshop Summary Contents     INTRODUCTION   1     WORKSHOP PROCEEDINGS   5     REFERENCES   35     APPENDIXES     A   GLOSSARY   37 B   PARTICIPANT BIOGRAPHIES   39 C   AGENDA   49

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