Bioscience/5. Molecular biology

Candidate of Agriculture Beyshova I.S.

Postgraduate student of natural sciences Kazimbekova T.B.

Kostanay State University named after A.Baytursynov, Republic of Kazakhstan

Different methods of DNA extraction from plant biological material

Extraction of the DNA of many plant species is considered difficult due to high concentration of secondary metabolites, such as polysaccharides and polyphenols, as well as due to proteins, fats and inhibitors in samples. In order to obtain highly purified DNA containing no inhibiting impurities it is necessary to use the most suitable extraction methods. There are several methods to solve this problem, also commercial kits are developed. Therefore, selection of optimal method for DNA extraction in an experimental way became necessary.

In our studies for selection of the optimal scheme of DNA extraction from fungi cultures we used the following methods:

- detergent-enzymatic method using dodecyl sulfate followed by extraction with phenol/chloroform;

- using the detergent Tween - 20 followed by extraction with phenol;

- using lysis buffer containing the detergent Nonidet P- 40 followed by extraction with phenol/chloroform;

- modified method using the detergent cetyl trimethyl ammonium bromide (CTAB).

The first method is based on the use of K proteinase enzyme for lysis of protein structures followed by phenol deproteinization.

The second method of DNA extraction from bacteria provides the use of Tween - 20 as detergent. Tween lyses cell membrane well but it has a weaker effect on the nucleus wall. Considering the fact that bacterial cell nucleus has no shell it is the best option.

The third and fourth methods involved Nonidet P- 40 and cetyl trimethyl ammonium bromide (CTAB) as detergents.

The method based on using CTAB (cetyl trimethyl ammonium bromide) as a detergent dissolves cell membranes well. Furthermore, it allows to divide DNA and polysaccharides, since they differ in solubility if the CTAB is present. At high salt concentrations (0.7M NaCl) nucleic acids form stable, but nevertheless soluble complexes with the CTAB. If the salt concentrations decrease below 0.4M NaCl CTAB/nucleic acid complexes set out, while the majority of polysaccharides remain in the solution.

The main criteria in developing optimal methods were concentration and purity of the preparation.

After Septoria fungus DNA was extracted using the above said methods qualitative and quantitative analysis of the sample was carried out. The ratio between the optical densities at 260 and 280 nm was measured spectrophotometrically. Maximum absorption of nucleic acids was recorded at 260 nm wave. The DNA preparation is considered to be free of impurities at the ratio E_260/280 equal to 1.8 and over. If this indicator is lower than the specified one, the sample is contaminated with proteins or phenol.

Samples of fungi culture DNA obtained using detergents Nonidet P- 40 and Tween - 80 were of poor quality. The ratio between the optical density at 260 and 280 nm waves averaged 1.65-1.7 that suggested DNA pollution with protein and other impurities. The results demonstrated the inefficiency of these methods.

The best results were obtained by treating mycelium suspension with detergent - cetyl trimethyl ammonium bromide (CTAB). Thus, the studies showed that optimal choice for us is a modified method of extracting genomic DNA from fungi culture using cetyl trimethyl ammonium bromide (CTAB) as the detergent.