Comparison of methods to preserve Rheum palmatum (Polygonaceae) for efficient DNA extraction and PCR amplification
Comparison of methods to preserve Rheum palmatum (Polygonaceae) for efficient DNA extraction and PCR amplification
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用于高效 DNA 提取和 PCR 扩增的鸡掌大黄（蓼科）保存方法比较
DOI：
10.4238/gmr.15038019
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发表时间：
2016-01-01
影响因子：
0.4
通讯作者：
Wang, X. M.
中科院分区：
文献类型：
--
作者：
Huang, M.;Sun, X. J.;Wang, X. M.
在本研究中，我们比较了用改良的CTAB法从14种不同方法保存的大黄叶中提取DNA的质量，包括在其他物种中常用的方法：在超冷（-80℃）温度下，用吸湿纸干燥后，用硅胶干燥，60℃干燥，70%乙醇，无水乙醇，70%乙醇添加50 mM EDTA， SDS-DNA提取液，核分离缓冲液，改进的NaCl-CTAB溶液，te -缓冲液，i -溶液或ii -溶液。以鲜叶提取的DNA作为对照。根据ITS2区域和微卫星标记的PCR扩增成功程度来评价提取DNA的质量。未从保存在核分离缓冲液和ii -溶液中的样品中提取DNA。超低温保存、70%乙醇和70%乙醇加50 mM EDTA保存、硅胶干燥和干燥后提取的DNA纯度高于其他方法保存的叶片，与新鲜叶片提取的DNA纯度相当。在本研究中，结合PCR扩增，超低温保存，硅胶干燥或干燥，以及PCR扩增提取的DNA可用于进一步的分子研究。
In this study, we compared the quality of DNA extracted using the modified CTAB method, from Rheum palmatum leaves preserved using fourteen different methods, including ones used commonly in other species: under ultra-cold (-80 degrees C) temperatures, after drying with an absorbent paper, desiccating using a silica gel, drying at 60 degrees C, in 70% ethanol, absolute ethanol, 70% ethanol supplemented with 50 mM EDTA, SDS-DNA extracting solution, nuclear separation buffer, improved NaCl-CTAB solution, TE-buffer, I-solution, or II-solution. DNA extracted from fresh leaves was used as the control. The quality of extracted DNA was evaluated based on the success of PCR amplification of the ITS2 region and a microsatellite marker. DNA was not extracted from samples preserved in the nuclear separation buffer and II-solution. The purities of DNA extracted from leaves preserved in ultra-cold temperatures, 70% ethanol, and 70% ethanol with 50 mM EDTA, and after desiccating using a silica gel and drying were higher, and comparable to the purity of DNA extracted from fresh leaves, than those of leaves preserved using other methods. In the present study, combined with the PCR amplifications, the preservation using ultra-cold temperatures, silica gel desiccation, or drying, and PCR amplification of the extracted DNA can be used for further molecular studies in R. palmatum.