Tamari Farshad, Hinkley Craig S, Ramprashad Naderia
Department of Biological Sciences, Kingsborough Community College, Brooklyn, New York 11235, USA.
J Biomol Tech. 2013 Sep;24(3):113-8. doi: 10.7171/jbt.13-2403-001.
Extraction of DNA from plant tissue is often problematic, as many plants contain high levels of secondary metabolites that can interfere with downstream applications, such as the PCR. Removal of these secondary metabolites usually requires further purification of the DNA using organic solvents or other toxic substances. In this study, we have compared two methods of DNA purification: the cetyltrimethylammonium bromide (CTAB) method that uses the ionic detergent hexadecyltrimethylammonium bromide and chloroform-isoamyl alcohol and the Edwards method that uses the anionic detergent SDS and isopropyl alcohol. Our results show that the Edwards method works better than the CTAB method for extracting DNA from tissues of Petunia hybrida. For six of the eight tissues, the Edwards method yielded more DNA than the CTAB method. In four of the tissues, this difference was statistically significant, and the Edwards method yielded 27-80% more DNA than the CTAB method. Among the different tissues tested, we found that buds, 4 days before anthesis, had the highest DNA concentrations and that buds and reproductive tissue, in general, yielded higher DNA concentrations than other tissues. In addition, DNA extracted using the Edwards method was more consistently PCR-amplified than that of CTAB-extracted DNA. Based on these results, we recommend using the Edwards method to extract DNA from plant tissues and to use buds and reproductive structures for highest DNA yields.
从植物组织中提取DNA往往存在问题，因为许多植物含有高水平的次生代谢产物，这些产物会干扰下游应用，如PCR。去除这些次生代谢产物通常需要使用有机溶剂或其他有毒物质对DNA进行进一步纯化。在本研究中，我们比较了两种DNA纯化方法：使用离子洗涤剂十六烷基三甲基溴化铵和氯仿 - 异戊醇的十六烷基三甲基溴化铵（CTAB）法，以及使用阴离子洗涤剂SDS和异丙醇的爱德华兹法。我们的结果表明，在从矮牵牛组织中提取DNA时，爱德华兹法比CTAB法效果更好。在八个组织中的六个组织中，爱德华兹法比CTAB法产生的DNA更多。在四个组织中，这种差异具有统计学意义，爱德华兹法产生的DNA比CTAB法多27 - 80%。在测试的不同组织中，我们发现开花前4天的花蕾DNA浓度最高，并且一般来说，花蕾和生殖组织比其他组织产生更高的DNA浓度。此外，使用爱德华兹法提取的DNA比CTAB法提取的DNA在PCR扩增中更稳定。基于这些结果，我们建议使用爱德华兹法从植物组织中提取DNA，并使用花蕾和生殖结构以获得最高的DNA产量。
