黄瓜DNA提取及其RAPD-PCR反应体系的优化

DNA Extraction and Conditions Optimization of Cucumber (Cucumis sativus L.) RAPD PCR

利用改良的 SDS方法提取黄瓜干种子与一步法提取的黄花苗总 DNA进行 RAPD扩增 ,其结果与传统的 CTAB和 SDS的结果一样。这两种方法不仅节约成本 ,且简单快速 ,大大降低了 DNA的提取难度 ,适宜黄瓜杂交种子纯度鉴定。不同组织材料对 RAPD无影响。研究了 RAPD的影响因素 ,改进及优化了 RAPD反应程序。结果表明 :Mg2 +浓度的范围为 1.5～ 3.0 mmol· L- 1 ,d NTPs浓度范围为 0 .15～ 0 .2 5 mmol· L- 1 ,Taq聚合酶浓度范围为 0 .5～ 1.5 U,模板DNA浓度为 2 0～ 10 0 ng,反应以及引物浓度为 0 .2～ 0 .4μmol· L- 1 。引物的碱基组成以及不同存放时间的引物对 RAPD扩增也有影响。 DNA预变性 94℃进行 4 min,94℃变性 30 s,37℃退火 30 s,72℃延伸 1min,循环 35次后

It was analyzed that methods to extract DNA template and different tissues had effects on RAPD. The RAPD products with total DNA extracted by improved SDS method from dry seeds and by 'one step' method from the yellowing young plants were the same as those with total DNA extracted by the traditional CTAB and SDS. Being simple and rapid, two methods could reduce the cost and make extraction of DNA very easily; thus they were suitable for the purity identification of Cucumber hybrid seeds. At the same time, it was shown that different tissues had no influence on RAPD. The paper also discussed the factors influenced RAPD and the results showed that the best Mg2+concentration for RAPD was 1.5-3.0 mmol.L -1 ; and the best dNTPs concentration was 0.15-0.25mmol.L -1 ; and suitable quantity of Taq polymerase was 0.5-1.5U; and suitable template DNA quantity was 20-100ng every reaction, and the best concentration of random primer was 0.2-0.4μmol.L -1 . In addition, it was further found that Taq polymerase with different factories and periods had effects on RAPD when the reaction system of RAPD was optimized, and that there were effects of random primers with different bases and periods on RAPD. The reaction pre denatured DNA at 94 ℃ for 4min, denatured DNA at 94 ℃ for 30sec, annealed primers at 37 ℃ for 30sec, extended primers at 72 ℃ for 1min, re extended reaction at 72 ℃ for 7min after 35 cycles and then stored the products at 4℃.