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Path to Effective Recovering of DNA from Formalin-Fixed Biological Samples in Natural History Collection: Workshop Summary Introduction APPLICATIONS OF DNA SEQUENCE INFORMATION In the past two decades, advancements in DNA-sequencing have opened many new doors that range from evolutionary biology to biomedical sciences to forensics. Taxonomists and systematists use genome analysis to decipher relationships within species and in the branching patterns of the tree of life (Eisen and Fraser, 2003). Diseases, such as breast and prostate cancer, can be caused by genetic alteration, and DNA sequencing allows the identiflcation of genetic biomarkers for those diseases (Rubin et al., 2002; King et al., 2003). Forensic scientists use nucleotide variants that are characteristic of an individual as the person’s identifler or DNA “flngerprint” (Gill et al., 1985). Taxonomists and systematists have developed another application for DNA sequencing: DNA barcoding is a technique for characterizing species of organisms using a short DNA sequence from a standard and agreed-upon position in the genome (CBOL, 2006). Barcoding could be a universally applicable diagnostic tool for species identiflcation. In fact, the Consortium for the Barcode of Life (CBOL), which is devoted to developing DNA barcoding as a global standard in taxonomy, has begun to construct a reference library that links species’ names with DNA barcode sequences (CBOL, 2006).
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Path to Effective Recovering of DNA from Formalin-Fixed Biological Samples in Natural History Collection: Workshop Summary NATURAL HISTORY COLLECTIONS AS A SOURCE OF DNA Natural history collections in museums and academic institutions contain a wealth of specimens that could be used to construct a DNA reference library. The specimens are invaluable; collectively, they constitute a partial documentation of biodiversity and they serve as the tools for evolutionary and comparative physiology and for many other disciplines. More important, many of those specimens are irreplaceable. Many natural history collection specimens are flxed and sometimes stored in formalin, which is inexpensive, widely available and effective, although it is an environmental toxin. A saturated solution of formaldehyde (CH2O) in water, formalin is about 37 percent formaldehyde by weight, and a standard flxation solution is 10 percent formalin in water, buffered to about pH 7. Formalin prevents degradation of specimens by microorganisms, and because it stabilizes and maintains the flne structure of soft tissue, it is still a widely used flxative. Aside from its toxic properties, formalin has another shortcoming—its use alters the DNA in samples. When they are exposed directly to formalin, mammalian cells undergo genetic and chromosomal alterations. Pathologists and other biomedical researchers who use archival tissue samples taken during epidemics, for example, or from victims of rare diseases have had some success in extracting DNA from formalin-flxed samples, but those samples have been embedded in paraffln rather than suspended in aqueous formalin or ethanol. Few of the many attempts to obtain and sequence DNA from formalin-flxed specimens stored in aqueous formalin or ethanol have been successful (Shedlock et al., 1997; Schander and Halanych, 2003). All of the protocols are slow, difflcult, and often expensive, and few produce DNA fragments longer than 500 base pairs. Development of an effective protocol for recovering DNA sequence information from specimens flxed in formalin and stored in formalin or alcohol will give access to sequence information for thousands of species that are extinct, rare, or difflcult to re-collect. WORKSHOP DESCRIPTION At the request of 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 Agriculture Research Service, and the U.S. Environmental Protection Agency’s Environmental Monitoring and
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Path to Effective Recovering of DNA from Formalin-Fixed Biological Samples in Natural History Collection: Workshop Summary Assessment Program, the National Research Council convened a one and a half day advanced workshop to discuss the future of DNA recovery from formalin-flxed specimens in museums or other natural history collections. The workshop’s participants were chemists, biophysicists, biochemists, molecular biologists, bioinformaticists, and researchers and managers of natural history collections interested in obtaining DNA from their specimens, all of whom participated actively. They examined attempts at DNA recovery on formalin-flxed specimens and discussed the research to advance the development of similar but more efflcient and cost-effective protocols (Box 1-1). BOX 1-1 Statement of Task for the Workshop on Recovering DNA from Formalin-Fixed Biological Samples The workshop was to bring together chemists, biophysicists, biochemists, geneticists, and bioinformaticians to examine past attempts on DNA recovery from formalin-preserved biological specimens and discuss the research needed to advance the development of similar but more efficient and cost-effective protocols. The goal of the workshop was to develop a research agenda that will shed new light on the problem and lead to a solution. Among the questions to be discussed at the workshop were: What is the state of preservation of DNA in the presence of formalin? Are the DNA chains intact or broken? Does formalin denature DNA or is it the process of extraction that is fragmenting the DNA? Are the nucleotides at each site being preserved or altered? How can the physical and chemical states of the DNA-formalin cross-linkages be better characterized? What additional information on these cross-linkages is needed? What new chemical and physical methods for DNA extraction should be tested, beyond those that have already been applied to formalin-fixed tissue? In what ways and to what extent can fragmented DNA be repaired physically and chemically after extraction from formalin? Can bioinformatics techniques be used to reconstruct the original sequence in silico from the DNA fragments recovered from formalin?
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Path to Effective Recovering of DNA from Formalin-Fixed Biological Samples in Natural History Collection: Workshop Summary Because it is known that formalin induces DNA fragmentation and nucleotide alteration, the workshop focused primarily on the extent and process of DNA damage and on DNA recovery from formalin-flxed samples stored in formalin or alcohol. The brieflng material distributed to participants specifled that some of the lessons learned from the protocol development for DNA recovery from formalin-flxed, paraffln-embedded biomedical specimens may be examined to assess whether they could be applicable to museum specimens. However, discussions on how to enhance protocols for extracting DNA from formalin-flxed, paraffln-embedded samples and discussions on extraction of ancient DNA were beyond the scope of the meeting. To plan the workshop, the National Research Council appointed a steering committee of experts in nucleic acid chemistry, structural chemistry, biomedical sciences, molecular biology, and biodiversity (Appendix B). The steering committee had several teleconferences to discuss the goals of the workshop with the sponsors, to identify workshop participants, and to discuss the workshop format. The workshop convened May 8-9, 2006, at the Keck Center of the National Academies. The participants were the steering committee members; representatives of sponsoring agencies; and others invited because of their expertise in biochemistry and biophysics of nucleic acids, organic chemistry, DNA repair proteins, optimization of DNA extraction, single-molecule sequencing, bioinformatics, mass spectrometry, DNA damage and repair, molecular biology, and taxonomy and systematics (Appendix B). The group included experts who could shed light on why DNA extraction from formalin-flxed samples had been mostly ineffective or unsuccessful, those who knew the methodologies for studying DNA damage, and “end users” who were attempting to obtain sequence information from formalin-flxed samples for their research. Participants discussed the questions outlined in the statement of task, and as a group developed a list of suggestions for how to move toward effective recovery of DNA from formalin-flxed samples in natural history collections.
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