环境样本中总DNA的提取及PCR条件的优化
DNA Extraction from Environmental Samples and Optimization of PCR Conditions
DOI: 10.12677/HJSS.2021.94023, PDF,   
作者: 吴玉清, 王永会*, 李 祥, 宋新贺：鸿灌环境技术有限公司，江苏 苏州 ；罗 辑：苏州市吴江生态环境局，江苏 苏州
关键词: DNA提取；纯度；PCR；优化；DNA Extraction； Pureness； PCR； Optimization
摘要: 本文用两种方法提取被污染河流底泥中的总DNA，比较两种方法的提取效果，研究环境样本DNA的PCR扩增的引物选择、对反应体系及反应程序进行相应的优化，得出适合PCR的最佳条件；研究表明氯仿-异丙醇提取法可以提取出含污染物质较少的总DNA；同时筛选出一些可扩增DNA模板的相关特异性引物；针对PCR扩增程序及体系都进行了一定程度上的优化。
Abstract: In this paper, two methods were used to extract the total DNA from the contaminated river sedi-ment, compare the extraction effect of the two methods, study the primer selection for PCR ampli-fication of environmental sample DNA, optimize the reaction system and reaction procedure ac-cordingly, and arrive at the best conditions for PCR; the study shows that the chloroform-isopropyl alcohol extraction method can extract the total DNA containing less contaminated material. The specific primers which can amplify DNA template were selected, and the PCR amplification proce-dure and system were optimized to some extent.
文章引用：吴玉清, 王永会, 李祥, 宋新贺, 罗辑. 环境样本中总DNA的提取及PCR条件的优化[J]. 土壤科学, 2021, 9(4): 176-185. https://doi.org/10.12677/HJSS.2021.94023

参考文献

[1] Courtois, S., Frotegard, A., Goransson, P., et al. (2001) Quantification of Bacterial Subgroups in Soil: Comparison of DNA Extracted Directly from Soil or Form Cells Previously Released by Density Gradient Centrifugation. Environ-mental Microbiology, 3, 43. [Google Scholar] [CrossRef] [PubMed]
[2] Ogram, A., Sayler, G.S., Barkey, T., et al. (1987) The Extraction and Purification of Microbial DNA from Sediments. Microbial, 7, 57-66. [Google Scholar] [CrossRef] 
[3] 宋培勇. 从土壤中提取DNA方法比较[J]. 微生物学杂志, 2006, 26(1): 109-112.
[4] Torsvik, V.L. and Goksoyr, J. (1978) Determination of Bacterial DNA in Soil. Soil Biology & Biochemistry, 10, 7-12. [Google Scholar] [CrossRef] 
[5] 朱灵峰, 张乐, 王海燕, 等. 河流沉积物中微生物DNA的提取方法比较研究[J]. 环境工程学报, 2011, 5(4): 935-938.
[6] Roose-Amsaleg, C.L., Gamier-Sillam, E. and Harry, M. (2001) Extraction and Purification of Microbial DNA from Soil and Sediment Samples. Applied Soil Ecology, 18, 47-60. [Google Scholar] [CrossRef] 
[7] 高平平, 赵立平. 可用于微生物群落分子生态学研究的活性污泥总DNA提取方法研究[J]. 生态学报, 2002, 22(11): 2015-2019.
[8] 赵勇, 周志华, 李武, 等. 土壤微生物分子生态学研究中总DNA的提取[J]. 农业环境科学学报, 2005, 24(5): 854-860.
[9] 奥斯伯金由辛, 包慧中, 赵丽云. 精编分子生物学实验指南[M]. 北京: 科学出版社, 2008.
[10] 黄婷婷, 曹慧, 王兴祥, 等. 一种土壤微生物总DNA的高效提取方法[J]. 土壤, 2004, 36(6): 662-666.
[11] 徐晓宇, 闵航, 刘和, 等. 土壤微生物总DNA提取方法的比较[J]. 农业生物技术,. 2005, 13(3): 377-381.
[12] 丁嫚, 李璐, 邹丽, 等. 活性污泥总DNA提取方法的比较[J]. 环境工程学报, 2009, 3(9): 1697-1702.
[13] 胡元森, 李翠香, 周毅, 等. 两种从土壤中提取DNA方法的比较生物技术[J]. 生物学报, 2006, 16(5): 34-36.
[14] Xing, D.E. and Godfrey, M.H. (2003) Nuclear DNA Analysis in Genetic Studies of Populations: Practice, Problems and Prospects. Molecular Ecology, 12, 563-584. [Google Scholar] [CrossRef] 
[15] Zhang, X., Yan, X., Gao, P., et al. (2005) Optimized Se-quence Retrieval from Single Bands of Temperature Gradient Gel Electrophoresis Profiles of the Amplified 16S rDNA Fragments from an Activated Sludge System. Journal of Microbiological Methods, 60, 1-11. [Google Scholar] [CrossRef] [PubMed]
[16] Qiu, X., Wu, L., Huang, H., et al. (2001) Evaluation of PCR-Generated Chimeras, Mutations and Heteroduplexes with 16S rRNA Gene-Based Cloning. Applied and Envi-ronmental Microbiology, 67, 880-887. [Google Scholar] [CrossRef] 
[17] Thompson, J.R., Marcelino, L.A. and Polz, M.F. (2002) Heteroduplexes in Mixed-Template Amplifications: Formation, Consequence and Elimination by “Reconditioning PCR”. Nucleic Acids Research, 30, 2083-2088. [Google Scholar] [CrossRef] [PubMed]
[18] 刘新春, 吴成强, 张昱, 等. PCR-DGGE法用于活性污泥系统中微生物群落结构变化的解析[J]. 生态学报, 2005, 25(4): 842-847.
[19] 闫晗, 吴祥云, 吴杏伟, 等. 土壤微生物16SrDNA V6 V8区PCR反应条件优化[J]. 应用化工, 2010, 39(9): 1325-1327.
[20] Watanabe, K., Kodama, Y. and Harayama, S. (2001) Design and Evaluation of PCR Primers to Amplify Bacterial 16S Ribosomal DNA Fragments Used for Community Fingerprinting. Journal of Microbiological Methods, 44, 253-262. [Google Scholar] [CrossRef] 
[21] 邸宁, 刘志民, 马焕普. 研究土壤微生物多样性的PCR条件优化[J]. 安徽农业科学, 2011, 39(23): 14059-14061.
[22] 李文红, 房振峰. 江蓠总DNA提取及其PCR扩增方法的建立[J]. 热带海洋学报, 2010, 29(6): 98-103.
[23] 杜涛, 黄小毛, 侯明生, 等. 从土壤中提取DNA用于PCR扩增[J]. 微生物学通报, 2003, 30(6): 1-6.
[24] 黄建华, 袁道强, 陈世峰. 生物化学实验[M]. 北京: 化学工业出版社, 2009: 2.

为你推荐