An Efficient, Economic, and Rapid Rice DNA Extraction Method and Its Application
A method for DNA extraction from rice leaf, root and seed was developed, and the extracted DNA was used as a template to successfully amplify the rice blast resistance gene Pita. Profiles of Pita in 165 breeding lines detected by DNA markers were verified using differential blast races. This method involved three steps: 1) Plant tissue was placed into a 200 μL 96-we!l plate; 2) A total of 70 μL of Buffer A containing NaOH and Tween 20 was added to each sample arid incubated at 95℃ for 10 min in a PCR machine; and 3) A total of 70 μL of Buffer B containing Tris-HCl and EDTA was added to each well and the resulting solution used for PCR amplification. Results demonstrated that this method of DNA extraction has the following advantages: 1) It is economical and only 4 common chemicals totaling 140 μL were used; 2) It is easy to perform, consists of only three steps, and one technician can extract hundreds of samples per day; 3) It requires a standard PCR machine; and 4) It can efficiently extract DNA from as little as a half dried seed, 5 to 20 mg of leaf tissue, or 20 mg root tissue. The resulting DNA quality was good enough to detect a single copy of the rice blast resistance gene Pi-ta which was verified in breeding lines with the expected disease reactions. This method has proven to be highly efficient for evaluating a large number of samples.
国家科技期刊平台
NSTL
万方数据
维普
