底泥水體中適用于PCR的不同形態DNA的提取方法

馮凌，羅義

南開大學環境科學與工程學院 環境污染過程與基準教育部重點實驗室，天津300071

底泥水體中適用于PCR的不同形態DNA的提取方法

馮凌，羅義*

南開大學環境科學與工程學院 環境污染過程與基準教育部重點實驗室，天津300071

提出直接從環境河流底泥和表層水體中同時提取不同形態DNA（胞內、胞外、染色體和質粒DNA）的有效方法.利用該方法提取了海河典型區域底泥的胞外DNA和胞內DNA及水體中細菌染色體DNA和質粒DNA.結果表明，NaH2PO4提取的胞外DNA分子量大于1kb，提取率40%～72%，沒有共提取胞內DNA.胞內DNA提取的分子量均大于23.1kb，細菌質粒DNA與染色體DNA同時分離.16S rDNA與sul2擴增驗證提取方法可靠性.16S rDNA擴增結果顯示所有胞外DNA呈陰性，胞內DNA呈陽性.質粒DNA和染色體DNA的16S rDNA擴增顯示，染色體DNA結果顯陽性，質粒DNA呈陰性，sul2的結果相反.

胞外DNA；胞內DNA；染色體DNA；質粒DNA；提取方法

Received 13 December 2009 accepted 10 January 2010

Abstract：In this paper,effective methods were presented to extract different gene types（extracellular DNA,intracellular DNA,genome DNA and plasmid DNA）from the sediments and water in environment.The methods were used for extracted extracellular DNA and intracellular DNA in sediments while genome DNA and plasmid DNA of bacteria in water in typical regions of Haihe River.Results showed that extracellular DNA was more than 1kb in size;its recovery ratio was 46%～72%without containing intracellular DNA by using NaH2PO4.All intracellular DNA were more than 23.1kb in size.Genome DNA and plasmid DNA were separated simultaneously.PCR amplification of 16S rDNA and sul2 were applied for assurance of the methods.Results showed that products of 16S rDNA of all the extracellular were negative while intracellular DNA positive.The genome DNA amplified of 16S rDNA turned out to be positive while of sul2 was negative,the results of plasmid DNA were the opposite.

Keywords：extracellular DNA；intracellular DNA；genome DNA；plasmid DNA；extraction

1 引言（Introduction）

近年來，抗生素的濫用導致在動物腸道細菌誘導產生抗生素抗性基因（Antibiotic resistance genes, ARGs），抗性基因從動物腸道細菌向環境土著細菌傳播擴散，使其成為一類新型環境污染物，其在不同環境介質中的傳播擴散目前已引起廣泛關注（Knapp et al.,2008;Krumperman,1983;Nielsen et al.,1998;2007;Pei et al.,2006;Pruden et al., 2006;羅義等，2008;高盼盼等，2009）.現代分子生物學PCR技術已被越來越多地用于環境樣本中抗性基因的檢測，如何獲得高質量的DNA模板是進行PCR擴增的前提.目前采用的環境樣本DNA的提取方法大都建立在對細胞內 DNA的提取上（Volossiouk et al.,1995;Clesceri et al.,1995）.然而，無論在陸地生態系統還是水生態系統都已檢測到胞外游離DNA的存在（Zhu,2006;Gallori et al.,1994;Poté et al.,2003）.許多研究表明，環境中包含持久性存在……