Path to Effective Recovering of DNA from Formalin-Fixed Biological Samples in Natural History Collections Workshop Summary (2006) / Chapter Skim
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Workshop Proceedings
Workshop Proceedings
Pages 5-34
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From page 5... ...
Biologists in various disciplines, for example, would like to use natural history specimens collected over 100-year spans for evolutionary, molecular, and genetic studies. However, users encounter major problems in obtaining both the DNA and the sequence information from those samples because of interference from the formalin fixation.
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From page 6... ...
suggested that, although the workshop's focus was on biological samples stored in aqueous solution, much could be learned from protocol development for DNA extraction from formalin-fixed and paraffin-embedded samples. Marvin Caruthers (University of Colorado)
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The process of formaldehyde fixation alters DNA in three ways: through fragmentation, sequence modification, and cross-linking. Cross-linking is not destructive to nucleic acids, and is reversible.
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That group reported that formalin fixation speeds sequence modification, but that the rate does not depend on the duration of formalin fixation. The ability to make a longer amplicon using polymerase chain reaction (PCR)
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agreed that sequence information could be obtained from nucleic acids reconstructed from fragments of depurinated DNA by single-molecule sequencing. However, Tom Evans (New England Biolabs, Inc.)
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Reliability of Sequence Information Some participants questioned the reliability of the sequence information obtained from formalin-fixed samples. For example, would certain DNA strands be more susceptible to sequence modification as a result of formalin fixation?
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Furthermore, hydroxyl damage has some minimal sequence preference, so lesions might not always occur in the same region. Variations in Curatorial Treatments Participants who work with natural history collections discussed the variations in the curatorial processing of biological samples.
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From page 12... ...
He mentioned that he chaired a task force at the National Cancer Institute to devise an optimal protocol for obtaining DNA from archival human tissues. That group reported that researchers in different laboratories use different protocols for sample preservation, and sometimes, even a slight variation can make a big difference in the success of DNA extraction.
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Cantor mentioned that the DNA sequences with multiple thymines in a row (called poly-T tracts) are more stable than others.
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From page 14... ...
Schindel said he would like to see the goldfish standard held in reserve until an acceptable approach to extraction protocol testing is developed. For example, Schindel had hoped that chemists could elucidate the degradation processes in formalin fixation that block DNA extraction or hamper PCR analysis, and then identify or develop better protocols.
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From page 15... ...
Because mass spectrometry is expensive, it is not used for standard sequencing. Rather, nucleic acids are sequenced by base-specific cleavage reactions.
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From page 16... ...
Similarly, base composition of DNA can be determined from the mass of the DNA strand and from the known masses of its adenine, guanine, thymine, and cytosine. The PCR products that Hofstadler typically examines are no more than 150 base pairs long.
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From page 17... ...
. Single-molecule sequencing requires fragmentation of genomic DNA but, as Hofstadler pointed out, that might not be necessary because the DNA obtained from formalin-fixed tissue is already fragmented.
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In Vitro Repair Evans discussed a "PreCR" method for in vitro DNA repair that involves treating damaged DNA in vitro with a mixture of DNA repair
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From page 19... ...
A broad spectrum of DNA damage is repaired by including other DNA repair enzymes that act in concert with the ligase and polymerase. At the time of the workshop, the PreCR enzyme mix Evans had been using could repair abasic sites, nicks, gaps, ultraviolet radiation damage, deaminated cytosine, and some forms of oxidative damage.
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From page 20... ...
of amplifiable DNA in formalin-fixed tissue than was available from fresh tissue. DNA yield varied slightly with the type of tissue used in the extraction.
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Second, the DNA extraction process itself could introduce PCR inhibitors. Mueller also mentioned that Sigma-Aldrich has been pursuing the development of a strand-based technique for DNA repair, similar to the one Evans presented.
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From page 22... ...
Therefore, he said, repairing DNA to obtain longer fragments might not be necessary, and identifying the samples with extractable DNA is more important than is refining extraction protocols. Evans countered that it is not known which aspect is the largest problem -- damage to DNA, inefficient DNA extraction, or the presence of PCR inhibitors -- in blocking retrieval of DNA sequence information from formalin-fixed samples.
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From page 23... ...
Ryan suggested having a set of samples that included different tissue types sent to different laboratories to test the same DNA extraction protocols. The outcome of systematic, simultaneous, independent testing of extraction protocols would provide information about protocol success, and whether some tissue types yield larger amounts of extracted DNA than others.
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From page 24... ...
Schander agreed that it would be worthwhile to increase depth of coverage to examine sequence modifications caused by formalin fixation. If sequence modification is induced by formalin, and if that is a common phenomenon, then the more that is known about it, the effects could be better predicted, said Hall.
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From page 25... ...
For example, some samples are stored in unbuffered formalin, others are fixed in formalin for different durations and some are transferred to ethanol after fixation. Because of those variations in curatorial treatment, the kinetics of formaldehyde and DNA reactions and the byproducts of different reactions are largely unknown.
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From page 26... ...
What new chemical and physical methods for DNA extraction should be tested, beyond those that have already been applied to formalinfixed tissue? Participants agreed that testing new methods for DNA extraction will not be fruitful if the condition of the DNA in formalin-fixed tissue is largely unknown because a failure to obtain sequence cannot be attributed unamibiguously to a failure of extraction protocol, or the absence of usable DNA in formalin-fixed samples, or the presence of PCR inhibitors.
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From page 27... ...
One complication is that formalin fixation could cause sequence modification and a sequence obtained from a formalin-fixed sample might not accurately represent the original sequence of the untreated sample. Whether formalin fixation introduces random or systematic error into a DNA sequence is not known, but it is worth investigating.
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From page 28... ...
The last phase would test how well the framework developed in the second phase could predict success in DNA extraction with a whole new set of specimens. To reveal practical limitations, O'Leary said, the process should be iterative and cover a spectrum of samples representing various species curatorial treatments.
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From page 29... ...
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From page 30... ...
Other factors could inhibit DNA extraction or PCR amplification, especially given the multiplicity of treatments used to preserve natural history specimens. Participants suggested designing a survey -- in consultation with experts at institutions that have natural history collections -- to gather information on curatorial history and on any successes or failures in the extraction of DNA or PCR amplification.
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From page 31... ...
· CloningPCRproductsforselectedformalin-fixedsamplesthathave a known gene sequence or an equivalent frozen or fresh sample could reveal whether formalin fixation induces sequence modification. A comparison of cloned PCR products from fixed tissue with products from fresh or frozen samples -- or with the known sequence -- would help to quantify mutations.
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From page 32... ...
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From page 33... ...
Ideas and suggestions from the workshop participants could further the efficient extraction of DNA from formalin-fixed samples, and that in turn could improve access to the sequence information of many rare or difficultto-collect species in natural history collections. Action by the Consortium for the Barcode of Life to follow up on the workshop participants' ideas and suggestions could facilitate the effective recovery of sequence information from formalin-fixed biological samples.
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