不同提取缓冲液对红树林土壤DNA提取品质的影响

doi:10.11978/j.issn.1009-5470.2011.02.087

热带海洋学报 ›› 2011, Vol. 30 ›› Issue (2): 87-93.doi: 10.11978/j.issn.1009-5470.2011.02.087cstr: 32234.14.j.issn.1009-5470.2011.02.087

不同提取缓冲液对红树林土壤DNA提取品质的影响

蒋云霞1, 郑天凌2, 3

1. 南方医科大学公共卫生与热带医学学院环境卫生学系, 广东广州 510515; 2. 厦门大学生命科学学院, 滨海湿地生态系统教育部重点实验室, 福建厦门 361005; 3. 厦门大学近海海洋环境科学国家重点实验室, 福建厦门 361005

收稿日期:2010-03-25 修回日期:2010-05-19 出版日期:2011-05-06 发布日期:2011-05-04
通讯作者: 郑天凌(1955—), 男, 福建省厦门市人, 教授, 博士生导师, 主要从事环境微生物研究。E-mail: wshwzh@xmu.edu.cn
作者简介:蒋云霞(1973—), 女, 湖北省武汉市人, 讲师, 博士, 主要从事环境微生物研究。E-mail: jiangyxia@yahoo.com.cn
基金资助:
国家高技术研究发展计划项目(2008AA09Z408);国家自然科学基金项目(40930847、30930017、30940002); 深港创新圈项目(08Lh-04); 南方医科大学公共卫生与热带医学学院院长基金项目(GW200801-039)

Evaluation of different extraction buffers on the quality of mangrove soil DNA

JIANG Yun-xia 1, ZHENG Tian-ling 2,3

1. Department of Environmental Health, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China; 2. Ministry of Education of Key Laboratory for Coast and Wetland Ecosystem, School of Life Sciences, Xiamen Uni-versity, Xiamen 361005, China; 3. State Key Laboratory for Marine Environmental Science, Xiamen University, Xiamen 361005, China

Received:2010-03-25 Revised:2010-05-19 Online:2011-05-06 Published:2011-05-04
Contact: 郑天凌(1955—), 男, 福建省厦门市人, 教授, 博士生导师, 主要从事环境微生物研究。E-mail: wshwzh@xmu.edu.cn
About author:蒋云霞(1973—), 女, 湖北省武汉市人, 讲师, 博士, 主要从事环境微生物研究。E-mail: jiangyxia@yahoo.com.cn
Supported by:
国家高技术研究发展计划项目(2008AA09Z408);国家自然科学基金项目(40930847、30930017、30940002); 深港创新圈项目(08Lh-04); 南方医科大学公共卫生与热带医学学院院长基金项目(GW200801-039)

摘要/Abstract

摘要：

针对1种典型的酸性、高有机质含量类型土壤——红树林土壤, 从DNA产量、DNA纯度、土壤腐殖酸类物质的提取量以及DNA提取物PCR(polymerase chain reaction)扩增反应的敏感度等方面比较了不同提取缓冲液对土壤DNA品质的影响。结果表明, 酸性SDS (sodium dodecyl sulfate)提取缓冲液所获得的土壤DNA产量及纯度均较低; PVP (polyvinylpyrrolidone)提取缓冲液所获得的土壤DNA产量较高, 但土壤腐殖酸类物质的提取量亦远高于其他提取缓冲液; 酸性SDS提取缓冲液及PVP提取缓冲液所获得的土壤DNA提取物即使稀释到10?3量级亦不能获得目的基因PCR扩增条带。PVPP (polyvinylpolypyrrolidone)提取缓冲液及CTAB (cetyltrimethylammonium bromide)+PVPP提取缓冲液所获得的土壤DNA产量较PVP提取缓冲液低, 但其纯度较高, 并且其纯度随提取缓冲液中PVPP浓度的升高而提高; 当2% PVPP提取缓冲液及CTAB+PVPP提取缓冲液所获得的土壤DNA提取物分别稀释到10?2及10?1量级时, 均能获得PCR扩增条带。综上所述, 酸性提取缓冲液及PVP提取缓冲液不适用于酸性、高有机质含量类型土壤DNA的提取, 而2% PVPP提取缓冲液及 CTAB+PVPP提取缓冲液均能提高此类土壤DNA的提取品质。

关键词: 红树林土壤, DNA提取, DNA产量, 腐殖酸, 提取缓冲液

Abstract:

The authors compared the influence of six different extraction buffers on DNA yields, DNA purity, co-extracted humic compound yields, and their PCR (polymerase chain reaction) sensitivity from a typical acidic and high organic content soil type, mangrove soil. Results showed that the quality of the soil DNA obtained using the acidic SDS (sodium dodecyl sul-fate) buffer was poor. The DNA yields obtained using the PVP (polyvinylpyrrolidone) buffer were higher than the other buff-ers, whereas the co-extracted humic compound yields were significantly higher. PCR results showed that no expected bands were obtained even in 1:1000 dilutions of the DNA extracts obtained using the acidic SDS buffer or PVP buffer. On the other hand, although the PVPP (polyvinylpolypyrrolidone) buffer and the CTAB (cetyltrimethylammonium bromide) and PVPP buffer resulted in a lower DNA yield, the purity of the soil DNA was improved. In addition, the purity was improved as the concentration of PVPP in the buffer increased. Moreover, PCR amplification was successfully conducted in the 1:10 dilutions of the DNA extracts obtained using the CTAB and PVPP buffer and in the 1:100 dilutions of DNA extracts obtained using the 2% PVPP buffer, respectively. In conclusion, acidic SDS buffer and PVP buffer were not suitable for extracting DNA from the acidic and high organic content soil type; however, 2% PVPP buffer and CTAB and PVPP buffer improved the DNA quality.

Key words: microbial diversity, DNA extraction, DNA yield, humic compound, extraction buffer

蒋云霞,郑天凌,. 不同提取缓冲液对红树林土壤DNA提取品质的影响[J]. 热带海洋学报, 2011, 30(2): 87-93.

JIANG Yun-xia ,ZHENG Tian-ling ,. Evaluation of different extraction buffers on the quality of mangrove soil DNA[J]. Journal of Tropical Oceanography, 2011, 30(2): 87-93.

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不同提取缓冲液对红树林土壤DNA提取品质的影响

Evaluation of different extraction buffers on the quality of mangrove soil DNA

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