Detection of crotamine and crotoxin gene sequences in genomic DNA from formaldehyde-fixed rattlesnakes

Abstract

Museum collections are an important source of material for studies of phylogeny, systematics, and phylogeography. These collections can represent rare or extinct species and provide historical samples and may be very useful for genetic studies of reptile populations. The material is usually formalin–fixed and stored in ethanol. Formalin (an aqueous solution of formaldehyde) is the fixative agent most widely used to preserve tissues and organisms, but it causes cross-links in DNA-DNA, DNA-protein and proteinprotein (Schander and Halanych 2003; Srinivasan et al. 2002). Several chemical reactions between formaldehyde and nucleic acids have been demonstrated, such as the addition of a hydroxymethyl group (-CH2 OH) in any nucleotide, formation of a methylene bridge between two amino groups, generation of apurinic and apyrimidic sites, and hydrolysis of phosphodiester bonds (Masuda et al. 1999; Srinivasan et al. 2002), causing the break of DNA, mutations and the inhibition of in vitro polymerization reactions of nucleic acids. The polymerase chain reaction (PCR) enzymatically amplifies specific sequences of nucleic acids, using very small amounts of DNA as template. In addition to innumerable current applications, PCR also allows nucleic acids from fixed tissues to be used in molecular analyses (Chatigny 2000; France and Kocher 1996; Gioia et al. 1998; Karlsen et al. 1994). Given that the Herpetological Collection Alphonse Richard Hoge at Instituto Butantan in São Paulo, Brazil has the largest Neotropical collection of snakes with a majority of Brazilian species, we decided to investigate if these formalin-fixed samples are useful for molecular analysis of venom variation. Thus, we studied the crotamine polymorphism of some Brazilian rattlesnakes. [...]