自动化工作站批量提取烤后烟叶 DNA 方法的建立与应用

摘要

To establish an efficient,rapid and accurate DNA extraction method from flue-cured tobacco,an automated method for flue-cured tobacco DNA extraction was developed by the BioMek NXP automation workstation combined with magnetic DNA purification kit.Results:Under conditions of water-bath heating 30 min,sample size 70~80 mg and 80% ethanol rinsing four times,OD260/OD280 ratio reached 1.7 ~ 1.9.Meanwhile,the qualification rate of DNA concentration above 100 ng/ L reached 95.8%.Tobacco NR gene was amplified well and no PCR inhibition was observed,which could be directly used in PCR qualitative analysis.DNA extraction of 96 samples could be finished in 110 min by this method.%为建立高效、快速、准确的烤后烟叶 DNA 提取方法，采用 BioMek NXP自动化工作站与国产DNA 磁珠法提取试剂盒相结合，建立了自动化批量提取烤后烟叶基因组 DNA（作为 PCR 模板）的方法，以获取高质量的烤后烟叶 DNA 满足 PCR 检测需求。结果表明：水浴时间30 min、样本量70～80 mg、80％乙醇洗涤4次能获得 OD260／OD280为1．7～1．9，浓度大于100 ng／μL 的烤后烟叶基因组 DNA，合格率达95．8％。经 PCR 定性扩增烟草硝酸还原酶基因（NR），该方法所提的 DNA 溶液中无 PCR 反应抑制物残留，可直接用于 PCR 定性分析。烟叶粉末经预处理，取上清液上机后约110 min 可完成96份样品的 DNA 提取工作。