摘要
DNA提取技术是分子实验的一个重要步骤,快速、高效的植物DNA提取方法是样品需要量大的分子实验的一个关键技术。本实验通过改进传统的DNA提取方法,使用96孔联体试管板,而不是单个离心试管,对叶片进行冷冻干燥后粉碎,无需使用液氮。在实验过程中,使用排枪操作,CTAB法提取DNA。以小麦为例,依据该方法提取幼苗叶片DNA,并选取6对引物对DNA样品进行PCR扩增,使用ABI PRISM3730 DNA分析仪对扩增产物进行分析,以检测提取的DNA质量。结果表明,本实验的DNA提取方法所得到的PCR扩增条带均有较高的强度。在6对实验引物的扩增结果中,扩增条带强度最好的引物有98%的样品在6935~16786 RFU（Relative Fluorescence Unit）之间;扩增条带强度较低的引物有93%的样品在532~1111 RFU之间;在所有的PCR扩增反应中,最低PCR扩增条带强度为205 RFU,缺带样品数量平均只有0.8%。按照本实验的方法操作,每人每天可提取上千个样品的DNA。因此,该DNA提取方法是一种高效、快速、能获得高质量DNA的有效方法。
DNA extraction is an important step in molecular assays.A rapid,high efficient method for extracting DNA is a major bottleneck for molecular studies requiring large amount of DNA samples.The traditional method was improved in this study.The plates of 96 connected tubes were used instead of separated tubes.The tissue was frozen and dried,then was ground instead of using liquid nitrogen.Multiple-pipettes and CTAB method were used to extract DNA for the method.The DNA of wheat seedling leaves was extracted according to this method as an example in this study.The DNA samples were amplified by 6 pairs of primers and PCR products were analyzed in an ABI PRISM 3730 DNA Analyzer for testing the quality of DNA samples.The results showed that the PCR products had high intensity.For the results amplified by the 6 pairs of primers,the highest range of intensity was 6 935～16 786 RFU in 98% of all tested samples,the lowest range of intensity was 532～1 111 RFU in 93% of all tested samples.The lowest intensity of all DNA samples amplified by all of the primers was 205 RFU.The average percentage of missing data was only 0.8%.According to this method,one person can extract about 1 000 DNA samples per day.Thus,the DNA extraction method in this study is a rapid,high efficient method for extracting high quality DNA.
出处
《麦类作物学报》
CAS
CSCD
北大核心
2011年第3期437-442,共6页
Journal of Triticeae Crops
基金
国家高技术研究发展计划(863计划)项目(2010AA101301)
国家外国专家局留学基金项目(CG2008320006)
