Scientia Marina, Vol 74, No 3 (2010)

DNA extraction from formalin-fixed tissue: new light from the deep sea

Ferran Palero
Evolutionary Genetics, Institute of Science and Technology Austria (ISTA), Austria

Sally Hall
National Oceanography Centre, Southampton, School of Ocean and Earth Science, University of Southampton, United Kingdom

Paul F. Clark
Department of Zoology, Natural History Museum, United Kingdom

David Johnston
Department of Zoology, Natural History Museum, United Kingdom

Jackie Mackenzie-Dodds
Department of Zoology, Natural History Museum, United Kingdom

Sven Thatje
National Oceanography Centre, Southampton, School of Ocean and Earth Science, University of Southampton, United Kingdom


DNA samples were extracted from ethanol and formalin-fixed decapod crustacean tissue using a new method based on Tetramethylsilane (TMS)-Chelex. It is shown that neither an indigestible matrix of cross-linked protein nor soluble PCR inhibitors impede PCR success when dealing with formalin-fixed material. Instead, amplification success from formalin-fixed tissue appears to depend on the presence of unmodified DNA in the extracted sample. A staining method that facilitates the targeting of samples with a high content of unmodified DNA is provided.


tetramethylsilane; ethidium bromide; formalin; Carcinus; Lithodidae

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Cawkwell, L. and P. Quirke. – 2000. Direct multiplex amplification of DNA from a formalin fixed, paraffin wax embedded tissue section. J. Clinic. Pathol. Mol. Pathol., 53: 51-52. doi:10.1136/mp.53.1.51 PMid:10884923    PMCid:1186903

Chang, Y. and G. Loew. – 1994. Reaction mechanisms of formaldehyde with endocyclic imino groups of nucleic acid bases. J. Am. Chem. Soc., 116: 3548-3555. doi:10.1021/ja00087a048

Chase, M., R. Etter, M. Rex and J. Quattro. – 1998. Extraction and amplification of mitochondrial DNA from formalin-fixed deep-sea mollusks. Biotechniques, 24: 243-247. PMid:9494724

Chaw, Y., L. Crane, P. Lange and R. Shapiro. – 1980. Isolation and identification of cross-links from formaldehyde-treated nucleic acids. Biochemistry, 19: 5525-5531. doi:10.1021/bi00565a010 PMid:7459328

Clark, P.F., M. Neale and P.S. Rainbow. – 2001. A morphometric analysis of regional variation in Carcinus Leach, 1814 (Brachyura: Portunidae: Carcininae) with particular reference to the status of the two species C. maenas (Linnaeus, 1758) and C. aestuarii (Nardo, 1847). J. Crustac. Biol., 21: 288-303. doi:10.1651/0278-0372(2001)021[0288:AMAORV]2.0.CO;2

Cunningham, C., N. Blackstone and L. Buss. – 1992. Evolution of king crabs from hermit crab ancestors. Nature, 355: 539-542. doi:10.1038/355539a0 PMid:1741031

Darling, J.A., M.J. Bagley, J. Roman, C.K. Tepolt and J.B. Geller. – 2008. Genetic patterns across multiple introductions of the globally invasive crab genus Carcinus. Mol. Ecol., 17: 4992-5007. doi:10.1111/j.1365-294X.2008.03978.x PMid:19120987

Diaz-Cano, S. and S. Brady. – 1997. DNA extraction from formalin-fixed, paraffin-embedded tissues: Protein digestion as a limiting step for retrieval of high-quality DNA. Diagn. Mol. Pathol., 6: 342-346. doi:10.1097/00019606-199712000-00006 PMid:9559294

Díaz-Viloria, N., L. Sánchez-Velasco and R. Pérez-Enríquez. – 2005. Inhibition of DNA amplification in marine fish larvae preserved in formalin. J. Plankton Res., 27: 787-792. doi:10.1093/plankt/fbi052

Fang, S., Q. Wan and N. Fujihara. – 2002. Formalin removal from archival tissue by critical point drying. Biotechniques, 33: 604-611. PMid:12238770

Folmer, O., M. Black, W. Hoeh, R. Lutz and R. Vrijenhoek. – 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotech. 3: 294-299. PMid:7881515

France, S. and T. Kocher. – 1996. DNA sequencing of formalin-fixed crustaceans from archival research collections. Mol. Mar. Biol. Biotech., 5: 304-313. PMid:8983199

García-Vázquez, E., P. Alvarez, P. Lopes, N. Karaiskou, J. Perez, A. Teia, J.L. Martinez, L. Gomes and C. Triantaphyllidis. – 2006. PCR-SSCP of the 16S rRNA gene, a simple methodology for species identification of fish eggs and larvae. Sci. Mar., 70S2: 13-21.

Gilbert, M.T.P., T. Haselkorn, M. Bunce, J.J. Sanchez, S.B. Lucas, L.D. Jewell, E. van Marck and M. Worobey. – 2007. The isolation of nucleic acids from fixed, paraffin-embedded tissues - which methods are useful when? PLoS ONE, 2(6): e537. doi:10.1371/journal.pone.0000537. doi:10.1371/journal.pone.0000537 PMid:17579711    PMCid:1888728

Hall, S. and S. Thatje. – 2009. Global bottlenecks in the distribution of marine Crustacea: temperature constraints in the family Lithodidae. J. Biogeogr., 36: 2125-2135. doi:10.1111/j.1365-2699.2009.02153.x

Huang, H. and P. Hopkins. – 1993. DNA interstrand cross-linking by formaldehyde: Nucleotide sequence preference and covalent structure of the predominant cross-link formed in synthetic oligonucleotides. J. Am. Chem. Soc., 115: 9402-9408. doi:10.1021/ja00074a005

Macpherson, E. – 1988. Revision of the family Lithodidae Samouelle, 1819 (Crustacea: Decapoda: Anomura) in the Atlantic Ocean. Mon. Zool. Mar., 2: 9-153.

Macpherson, E. – 2003. Some lithodid crabs (Crustacea: Decapoda: Lithodidae) from the Solomon islands (SW Pacific Ocean) with the description of a new species. Sci. Mar., 67: 413-418.

Martín Andrés, A., A. Silva Mato, J.M. Tapia García and M.J. Sánchez Quevedo. – 2004. Comparing the asymptotic power of exact tests in 2x2 tables. Comp. Stat. & Data Anal., 47(4): 745-756. doi:10.1016/j.csda.2003.11.012

McLaughlin, P. – 1983. Hermit crabs - are they really polyphyletic? J. Crustac. Biol., 3: 608-621. doi:10.2307/1547956

Palero, F., K.A. Crandall, P. Abelló, E. Macpherson and M. Pascual. – 2009. Phylogenetic relationships between spiny, slipper and coral lobsters (Crustacea: Decapoda: Achelata). Mol. Phylogenet. Evol., 50: 152-162. doi:10.1016/j.ympev.2008.10.003 PMid:18957325

Quay, W. – 1974. Bird and mammal specimens in fluid-objectives and methods. Curator, 17: 91-104. doi:10.1111/j.2151-6952.1974.tb01228.x

R Development Core Team. – 2009. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL

Saiki, R., D. Gelfand, S. Stoffel, S. Scharf, R. Higuchi, G. Horn, K. Mullis and H. Erlich. – 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239: 487-491. doi:10.1126/science.2448875 PMid:2448875

Sambrook, J., E. Fritsch and T. Maniatis. – 1989. Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, New York.

Santos, S., D. Sá, E. Bastos, H. Guedes-Pinto, I. Gut, F. Gärtner and R. Chaves. – 2009. An efficient protocol for genomic DNA extraction from formalin-fixed paraffin-embedded tissues. Res. Vet. Sci., 86: 421-426. doi:10.1016/j.rvsc.2008.08.007 PMid:18926550

Scatena, M. and E. Morielle-Versute. – 2008. Suitability of DNA extracted from archival specimens of fruit-eating bats of the genus Artibeus (Chiroptera, Phyllostomidae) for polymerase chain reaction and sequencing analysis. Gen. Mol. Biol., 31: 160-165. doi:10.1590/S1415-47572008000100027

Schander, C. and K. Halanych. – 2003. DNA, PCR and formalinized animal tissue - a short review and protocols. Org. Diver. Evol., 3: 195-205. doi:10.1078/1439-6092-00071

Shi, S., R. Datar, C. Liu, L. Wu, Z. Zhang, R. Cote and C. Taylor. – 2004. DNA extraction from archival formalin-fixed, paraffin-embedded tissues: Heat-induced retrieval in alkaline solution. Histochem. Cell Biol., 122: 211-218. doi:10.1007/s00418-004-0693-x PMid:15322858

Shibata, D. – 1994. Extraction of DNA from paraffin-embedded tissue for analysis by polymerase chain-reaction - new tricks from an old friend. Human Pathol., 25: 561-563. doi:10.1016/0046-8177(94)90219-4

Simmons, J. – 1995. Storage in fluid preservatives. In: C. Rose, C. Hawks and H. Genoways (eds.), Storage of natural history collections: A preventive conservation approach, pp. 161-186. Society for the Preservation of Natural History Collections, Iowa.

Takeda, M. and Y. Ohta. – 1979. A new species of Lithodidae from Suruga Bay, central Japan. Bull. Natl. Sci. Mus. A, 5: 195-200.

Thatje, S., K. Anger, J. Calcagno, H. Lovrich, H. Pörtner and W. Arntz. – 2005. Challenging the cold: Crabs reconquer the Antarctic. Ecology, 86: 619-625. doi:10.1890/04-0620

Thatje, S., S. Hall, C. Hauton, C. Held and P.A. Tyler. – 2008. Encounter of lithodid crab Paralomis birsteini on the continental slope off Antarctica, sampled by ROV. Polar Biol., 31: 1143-1148. doi:10.1007/s00300-008-0457-5

Ubero-Pascal, N., J. Fortuño and M. Puing. – 2005. New application of air-drying techniques for studying Ephermeroptera and Plecoptera eggs by scanning electron microscopy. Micr. Res. Tech., 68: 264-271. doi:10.1002/jemt.20248 PMid:16315231

Zaklan, S. – 2002. Review of the family Lithodidae (Crustacea: Anomura: Paguroidea): Distribution, biology and fisheries. In: A. Paul, E. Dawe, R. Elner, G. Jamieson, G. Kruse, R. Otto, B. Sainte-Marie and T. Shirley (eds.), Crabs in cold water regions: Biology, management and economics, pp. 58-84. Alaska Sea Grant College Program, Fairbanks.

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