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作 者 ：胡雪华;陈香;肖宜安*;廖信军;鞠建文;郭永久

期 刊 ：植物研究 2011年 31卷 1期 页码：89-94

关键词：RAPD；反应体系；优化；血水草；

Keywords:RAPD, reaction system, optimization, Eomecon chionantha Hance,

摘 要 ：为建立血水草的RAPD-PCR最佳反应体系，对影响血水草RAPD反应的Mg²⁺、模板DNA、dNTPs、引物浓度和Taq聚合酶用量等因素进行优化。结果表明：25 μL反应体系中含10×Buffer 2.5 μL，1.8 mmol·L^-1 Mg²⁺，2U Taq DNA聚合酶，50 ng模板，0.2 mmol·L^-1 dNTPs，1.6 μmol·L^-1引物。扩增程序为：94℃预变性2 min；预扩增：94℃变性20 s，36℃退火30 s，72℃延伸75 s，5个循环；扩增：94℃变性20 s，40℃退火30 s，72℃延伸60 s，40个循环；72℃保温20 min，4℃保存。所建立的血水草RAPD-PCR反应体系具有标记位点清晰、反应系统稳定、检测多态性能力强、重复性好等特点，可以较好地应用于血水草的遗传多样性分析研究。

Abstract:In order to establish the optimal reaction of RAPD-PCR amplification for Eomecon chionantha Hance, the following parameters were optimized: the concentrations of Mg²⁺, template DNA, dNTPs, primers and Taq DNA polymerase. The optimal RAPDPCR reaction system was as follows a total volume of 25 μL contains 2.5 μL 10×Buffer, 1.8 mmol·L^-1 Mg²⁺，2 U Taq DNA polymerase, 50 ng template DNA, 0.2 mmol·L^-1 dNTPs and 1.6 μmol·L^-1 primer. The reaction program fitting to the RAPD-PCR was as follows: initial denaturation at 94℃ for 2 min, followed by 5 beforehand cycles of 94℃ for 20 s, 36℃ for 30 s, 72℃ for 75 s; followed by 40 cycles of 94℃ for 20 s, 40℃ for 30 s, 72℃ for 60 s, and a final exposure to 72℃ for 20 min, then stored in 4℃. This RAPD-PCR system has some distinguising features including clear marker site, stable reaction system, reliable abundant polymorphisms, and better repeatability. It is suitable for the study on genetic diversity of E.chionantha.

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