Klanten, Selma O., Van Herwerden, Lynne, and Choat, J. Howard (2003) Acquiring reef fish DNA sequences from formalin-fixed museum specimens. Bulletin of Marine Science, 73 (3). pp. 771-776.

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Abstract

In recent years molecular techniques have invaded the marine realm. These techniques are mainly used to determine phylogenetic relationships between species, and for population genetic and phylogeographic studies. The use of museum specimens is advantageous, especially for phylogenetic studies. It enables sampling of species that are extinct or endangered, and those that are difficult to obtain fresh from the wild. Such specimens may be quite old (from collections at the beginning of the 20th century) and are generally fixed in formalin (10%) prior to being preserved in alcohol. The general consensus is that formalin-fixed tissue cannot be used for DNA studies, as yields are very low and DNA is substantially degraded (Dillon et al., 1996; Wirgin et al., 1997). However, a few studies described and compared DNA extractions from different preservation techniques, including formalin-fixed specimens of tapeworms (Li et al., 2000), trout (Shiozawa et al., 1992), Atlantic coast bass (Wirgin et al., 1997), molluscs (Chase et al., 1998), a range of other taxa (amphibian, reptile, fish, invertebrate; Shedlock et al., 1997), and insects (Dillon et al., 1996). All of these studies, with the exception of the last one, successfully extracted mitochondrial DNA from formalin-fixed samples. The protocol we developed to extract DNA from mitochondrial and nuclear DNA from museum collections of reef fish, focused on the genera Naso (Family: Acanthuridae), Scams and Chlorurus (Family: Scaridae). Here we present this method for DNA extractions from formalin-fixed reef fish specimens, subsequent PCR (polymerase chain reaction) amplification, and sequencing of such samples. The technique described here differs from the above studies in its detailed methodology, including extraction time and chemicals used. Furthermore, it compares the differences for PCR optimization and amplification between ethanol-preserved (fresh) and formalin-fixed tissues. This study is the first to document successful sequencing of a nuclear marker from formalin-fixed samples.

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