Abstract
DNA extraction of plants with high quality is very important to researches in molecular biology. Several extraction protocols have been used to obtain soybean DNA; however, there is a lack of papers about extraction protocols optimization and the best developmental stage of the plant to collect them. Therefore, the main purpose of the study was to extract high quantity and quality of DNA from fresh or frozen soybean samples, using different protocols. Moreover, we analyzed the best developmental stage of the plant to do the extraction. Fresh leaves or leaves kept for two years in the ultra-freezer were submitted to the DNA extraction protocols: Haberer et al . , 1996 (modified); second modification from Haberer et al., 1996; Murray & Thompson, 1980 (modified) e Doyle & Doyle, 1990 (modified). Modified protocol of Doyle & Doyle was used to value the best stage to collect the leaves to do the DNA extraction. The samples were collected in the stages of development VC, V1, V2, V3, V4 and R5. The experiments were conducted in completely randomized design with 10 samples per treatment. The data underwent variance analysis and the averages were compared by the Tukey test (p<0.05). Through Doyle & Doyle, 1990 and Haberer et al., 1996 modified protocols, for both fresh and frozen samples, it was possible to obtain a higher total DNA concentration if compared to the other tested protocols. However, the quality of DNAs extracted by the protocol Doyle & Doyle, 1990 (modified) was superior, due to a minor molecular degradation. Besides that, the extractions made with these protocols have shown to be more efficient using frozen leaves’ tissue. Higher DNA concentrations were obtained analyzing VC samples; however, there were no statistical differences between the stages VC, V2 and V3. It is suggested thereby to use modified of Doyle & Doyle for DNA extraction from soybean leaves in V2 and V3 stages of development from frozen samples, providing the collect of a large number of samples and its storage until the analysis.
Highlights
DNA extraction of plants with high quality is important to researches in molecular biology (CANKAR et al, 2006)
Vegetal DNA preparations are, commonly, used as substrates in Polymerase Chain Reactions (PCR) for phylogenetic studies or in the development of molecular markers, such as the microsatellites and the ones generated by Random Amplified Polymorphic DNA (RAPD)
Most protocols described in the literature uses the CTAB standard protocol, with some modifications in order to solve specific problems from the specie in study (ROMANO; BRASILEIRO, 1999)
Summary
DNA extraction of plants with high quality is important to researches in molecular biology (CANKAR et al, 2006). A lot of problems are described about its isolation and purification (MAZZA; BITTENCOURT, 2000). These obstacles are due to the high content of proteins, polysaccharides and secondary metabolites, like phenolic compounds, that can be extracted jointly with the DNA, affecting its quality (MALIYAKAL, 1992). The DNA extraction method with most success for different species is the one based on the reagent CTAB (Cetyl Trimethylammonium Bromide). This detergent solubilizes membranes, forming a complex with the DNA that facilitates the posterior precipitation of the DNA molecule. Most protocols described in the literature uses the CTAB standard protocol, with some modifications in order to solve specific problems from the specie in study (ROMANO; BRASILEIRO, 1999)
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