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棉花学报 ›› 2016, Vol. 28 ›› Issue (4): 413-417.doi: 10.11963/issn.1002-7807.201604014

• 研究简报 • 上一篇    下一篇

改进的TPS法:一种棉花叶片DNA快速提取方法

张友昌,冯常辉,别墅,王小娇,易先达,张成,秦鸿德*   

  1. 湖北省农业科学科院经济作物研究所,湖北 武汉 430064
  • 收稿日期:2015-08-21 出版日期:2016-07-15 发布日期:2016-07-15
  • 通讯作者: 副研究员,qinhongde2002@163.com
  • 作者简介:张友昌(1984―),男,硕士,zhych307@163.com
  • 基金资助:
    湖北省农业科学院青年科学基金(2015NKYJJ19)

An Improved TPS Method for Rapid DNA Extraction from Cotton Leaves

Zhang Youchang, Feng Changhui, Bie Shu, Wang Xiaojiao, Yi Xianda, Zhang Cheng, Qin Hongde*   

  1. Cash Crop Institute, Hubei Academy of Agricultural Sciences, Wuhan, Hubei 430064, China
  • Received:2015-08-21 Online:2016-07-15 Published:2016-07-15

摘要: 分子标记辅助选择需要大批量的DNA样品,快速提取DNA的方法是提高选择效率的关键。研究比较多种植物DNA的提取方法,通过优化改进,建立了适于棉花叶片基因组微量快速提取的新方法。该方法增加缓冲液预处理及在TPS缓冲液中添加PVP40,采用PCR板批量取样、微型手电钻磨样,省去了蛋白抽提、DNA沉淀、洗涤、风干、溶解等步骤,免去氯仿和异戊醇的使用。本方法具有低成本、快速、高通量、操作简单等特点,而且能确保提取的棉花DNA的浓度和纯度达到要求,满足大批量样本SSR分子标记辅助育种工作的需要。

关键词: 棉花; DNA; 快速提取; CTAB法; SSR

Abstract: An improved method of rapid simple DNA extraction from cotton leaves was established based on the TPS (named by using TPS buffer—a mixture of Tris-HCl, EDTA and KCl) procedure to increase the efficiency of molecular marker-assisted selection. Compared with cetyltrimethyl ammonium bromide (CTAB) and TPS procedures, this new method is superior as it uses fewer reagents, and is simpler, faster, and less expensive. This new method makes use of a pre-buffer with 2% (mass fraction) PVP40 added to the TPS buffer (0.1 mol·L-1 Tris-HCl, 0.01 mol·L-1 EDTA, 1.2 mol·L-1 KCl), a 96-well PCR plate, and a miniature hand drill to grind samples. This new method could omit protein extraction, DNA precipitation, washing, drying, and dissolving procedures, and does not require chloroform or isoamyl alcohol. Using this method, more than 600 DNA samples can be extracted in a day by one person, and the extracted DNA is suitable for SSR (simple sequence repeat) analysis. Thus, the method can be used in cotton molecule marker-assisted selection.

Key words: cotton; DNA; rapid extraction; cetyltrimethyl ammonium bromide method; simple sequence repeat

中图分类号: 
  • S562.035