Comparison and improvement of DNA extraction methods in Buxus hyrcana

Abstract

Buxus hyrcana is one of the endangered and evergreen species of the Hyrcanian forests in Iran. The genetic diversity assessment is an essential step towards the conservation of this species. High-quality DNA is required for molecular markers analysis; therefore we compared different DNA extraction methods on leaf samples of B. hyrcana. The quantity and quality of the extracted DNAs were evaluated by spectrophotometry and gel electrophoresis. Also, ISSR (Inter simple sequence repeats) markers were applied on the extracted DNAs to compare their quality for PCR amplification. Results showed that quantity, quality, and PCR efficiency and reproducibility were different for DNA extracted using different methods. The quality of the DNA at the absorbance A260/A280 ratio ranged from 1.02 to 1.97. The highest concentration of DNA measured by spectrophotometry belonged to the Cota-Sanchez extraction protocol (695.3 ng/l) and the lowest value was obtained with Edward4 method (204.7 ng/l). The modified Onate method (Onate2) was extracted the highest DNA concentration by comparison of brightness against the DNA ladder. Among the different extraction methods, the good quality and quantity were obtained in extracted DNA for Doyle and Doyle, Cota-Sanchez and modified Onate protocols; the latter method (Onate2) created both good quality and quantity of extracted DNA and operated effectively in terms of cost and time. Onate2 had the best amplification results with ISSR primers.