Print version ISSN 0100-8455
Braz. J. Genet. vol.20 no.3 Ribeirão Preto Sept. 1997
Extraction of genomic DNA from Melipona quadrifasciata (Hymenoptera: Apidae, Meliponinae)
Ana Maria Waldschmidt, Tânia Maria Fernandes Salomão, Everaldo Gonçalves de Barros and Lúcio de Antônio Oliveira CamposDepartamento de Biologia Geral, Universidade Federal de Viçosa (UFV),
36571-000 Viçosa, MG, Brasil. Send correspondence to A.M.W.
The objective of the present study was to test three different procedures for DNA extraction of Melipona quadrifasciata based on existing methods for DNA extraction of Apis, plants and fungi. These methods differ in the concentrations of specific substancesin the extraction buffer. The results demonstrate that the method used for Apis is not adequate for DNA extraction from M. quadrifasciata. On the other hand, with minor modifications this method and the methods for plants and fungi were adequate for DNA extraction of this stingless bee, both for adults and larvae.
Molecular markers (RAPD, RFLP, AFLP, etc.) have being widely usedin plant and animal breeding as well as for the elucidation of molecular aspects of evolution, phylogeny and physiology.
These techniques require a fair amount of DNA of good quality. This is a particularly serious problem with small animals like insects. In these cases, specific methodologies to optimize the extraction of good quality DNA samples are needed.
Few methods are available for beeDNA extraction in the literature. To our knowledge, only Sheppard and McPheron (1991) and Hunt and Page (1992) have described DNA extraction procedures for the genus Apis. In this work three DNA extraction methodologies used in different systems were modified and tested with Melipona quadrifasciata, a stingless bee popularly known in Brazil as “mandaçaia”.
MATERIAL AND METHODS
Fifteen adultworkers and 15 larvae from the same colony were used for DNA extraction of Melipona quadrifasciata. Three different methods were tested: A) method described by Doyle and Doyle (1990) for plants and by Schäfer and Wöstemeyer (1992) for fungi; B) method described by Hunt and Page (1992) for Apis, and C) modified method B.
Basically, the three methods tested differ in the concentrations of specificcomponents of the extraction buffer. In method A, the extraction buffer was 100 mM Tris-HCl, pH 8.0, containing 2% CTAB, 1.4 M NaCl, 20 mM EDTA and 100 g/ml proteinase K. In B, the extraction buffer was 50 mM Tris-HCl, pH 8.0, containing 1% CTAB (hexadecyl trimethyl ammonium bromide), 0.75 M NaCl, 10 mM EDTA and 100 g/ml proteinase K. In C, the extraction buffer was 50 mM Tris-HCl, pH 8.0,containing 2% SDS (sodium dodecyl sulfate), 0.75 M NaCl, 10 mM EDTA and 100 g/ml proteinase K.
Each adult bee was ground individually in a mortar and pestle containing liquid N2 and homogenized with extraction buffer. Each larva was ground in a microcentrifuge tube with a plastic pestle in the presence of extraction buffer. The samples were then incubated at 65oC for 30 min. Samples containing adultindividuals were deproteinized twice with one volume each of chloroform. Larva homogenates were then deproteinized once with one volume phenol:chloroform (1:1) and once with one volume chloroform. After deproteinization the samples were centrifuged at 11,750 g for 5 min. Nucleic acid was precipitated by addition of one volume of cold isopropanol and incubation at -20oC for 2 to 24 h. Aftercentrifugation at 16,000 g for 30 min the pellets were washed twice with 70% (v/v) ethanol and vacuum dried for approximately 15 min or dried in the air for 30 min. The pellet was resuspended in 200 l TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and incubated with RNAse A (100 g/ml) for 30 min at 37oC. The integrity and purity of the DNA samples were checked on 0.8% (w/v) agarose mini-gels.