Selection and optimization of DNA extraction methods from the leaves of Gleditsia triacanthos L.

Abstract

Abstract. Currently, molecular genetic methods using DNA markers are increasingly used in studies of polymorphism of various populations of woody and shrubby plants. The purpose of this work was the evaluation and selection of protocols for the isolation and purification of DNA from the leaves of Gleditsia triacanthos L. for further studies using DNA labeling. Methods. Four protocols were used to isolate DNA from the leaf blade of Gleditsia triacanthos L. Anionic detergent sodium dodecyl sulfate was used in three isolation protocols for cell lysis, potassium acetate was used for purification from polysaccharides and proteins. In the fourth protocol, a cationic surfactant cetyltrimethyl ammonium bromide was used for cell lysis, the extract was purified with a mixture of chloroform-isoamyl alcohol (24 : 1). Precipitation of the isolated DNA was carried out with isopropanol. The quality of the isolated DNA was evaluated by spectrophotometry, horizontal electrophoresis and Real-time PCR with two types of primers. Results. Optimal conditions for DNA extraction from samples of Gleditsia triacanthos L. containing a large number of metabolites affecting the quality of the isolated extract were selected. By electrophoresis, it was found that both the isolation protocol with sodium dodecyl sulfate and the isolation protocol with cetyltrimethyl ammonium bromide make it possible to obtain a sufficient amount of DNA. The most purified DNA was obtained by the third protocol using sodium dodecyl sulfate and dithiotreitol and by the fourth protocol using cetyltrimethylammonium bromide. The results of PCR of the obtained samples with ITS and psbI-psbK primers indicate that a sufficient amount of product has been obtained and the reproducibility of ISSR markers. The scientific novelty of the work consists in choosing the optimal method of DNA extraction from the leaves of Gleditsia triacanthos L., which is a complex object containing a large number of potential PCR inhibitors. The protocol with sodium dodecyl sulfate and dithiotreitol made it possible to obtain DNA in the right amount and of acceptable quality.