Detection of Crotamine and Crotoxin Gene Sequences in DNA of Formaldehyde Fixed Rattlesnakes.

Corrêa, P. G.¹; Germano, V. J.¹; Rádis-Baptista, G.^2,3; Yamane, T.²; Oguiura, N.¹

1 Lab. de Herpetologia e  2 Lab. de Toxinologia Molecular, Instituto Butantan, São Paulo, Brazil; 3 Atualmente no Depto. de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, Ceará, Brazil

PCR is a useful tool for the DNA analysis since it is possible to detect sequences starting with a small quantity of nucleic acids and the integrity of DNA is not essential for the amplification of the sequence of interest. The Instituto Butantan has the greatest collection of Neotropical snakes in the world with 80000 specimens. The voucher snakes are fixed in 10% formaldehyde, washed in tap water and stored in 70% ethanol. Since we work on South American rattlesnake toxins, the crotamine and crotoxin genes were used as the model to study the possibility of using DNA of fixed snakes in PCR experiments. The DNA was purified from fixed tissues using proteinase K and SDS followed by phenol / chloroform extraction and analyzed in agarose electrophoresis. We observed that the genomic DNAs were broken in fragments of about 500 bp. Despite the DNA degradation it was possible to amplify the crotoxin gene sequence using DNA from tissues tested and the crotamine gene sequence using DNA from crotamine-positive rattlesnake tissues. These results show that tissues from formaldehyde fixed snakes can be used as material in PCR analysis of DNA.

Supported by FAPESP, Fundap.

Correspondence to: pgcorrea@ig.com.br or nancyoguiura@butantan.gov.br