Polymerase chain reaction amplification of naphthalene-catabolic and 16S rRNA gene sequences from indigenous sediment bacteria
Polymerase chain reaction amplification of naphthalene-catabolic and 16S rRNA gene sequences from indigenous sediment bacteria
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本地沉积物细菌萘分解代谢和 16S rRNA 基因序列的聚合酶链式反应扩增
DOI：
10.1128/aem.59.3.687-694.1993
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发表时间：
1993
影响因子：
4.4
通讯作者：
W. Ghiorse
中科院分区：
文献类型：
--
作者：
J. B. Herrick;E. Madsen;C. Batt;W. Ghiorse
我们报告了通过聚合酶链反应（PCR）从未接种的表面和地下沉积物中扩增细菌基因。当将含有大约 10(7) 个细胞的地下沉积物直接添加到 PCR 混合物中时，本地细菌 16S 核糖体 DNA 基因的 PCR 扩增不成功。然而，当10mg沉淀物接种约10(5)个恶臭假单胞菌G7细胞时，通过PCR扩增检测到恶臭假单胞菌G7 NAH7质粒特征性的nahAc萘双加氧酶基因。 PCR 扩增产物的 Southern 印迹提高了对接种恶臭假单胞菌 G7 的样品中的 10(3) 至 10(4) 细胞的敏感性，但未添加沉淀物的对照显示 PCR 被沉淀物部分抑制。在聚乙烯吡咯烷酮存在下，将溶菌酶-十二烷基硫酸钠冻融 DNA 提取与凝胶电泳部分纯化相结合，以使用真细菌 16S 核糖体 DNA 引物通过 PCR 扩增来自表面或地下沉积物样品中的本地细菌的 DNA。当通过增加 Taq 和引物浓度来修改 PCR 条件时，还可以使用 nahAc 特异性引物从本地细菌中扩增 nahAc 基因。来自地表和地下沉积物的 nahAc 扩增产物的限制性消化揭示了相对于恶臭假单胞菌 G7 的多态性。本文描述的 DNA 提取、纯化和 PCR 扩增程序表明，PCR 是研究沉积物和地下水环境中本土微生物群落的功能和分类特异性 DNA 的潜在有用工具。
We report the amplification of bacterial genes from uninoculated surface and subsurface sediments by the polymerase chain reaction (PCR). PCR amplification of indigenous bacterial 16S ribosomal DNA genes was unsuccessful when subsurface sediment containing approximately 10(7) cells.g-1 was added directly to a PCR mixture. However, when 10 mg of sediment was inoculated with approximately 10(5) cells of Pseudomonas putida G7, the nahAc naphthalene dioxygenase gene characteristic of the P. putida G7 NAH7 plasmid was detected by PCR amplification. Southern blotting of the PCR amplification product improved sensitivity to 10(3) to 10(4) cells from samples inoculated with P. putida G7, but controls with no sediment added showed that the PCR was partially inhibited by the sediments. Lysozyme-sodium dodecyl sulfate-freeze-thaw DNA extraction was combined with gel electrophoretic partial purification in the presence of polyvinylpyrrolidone to render DNA from indigenous bacteria in surface or subsurface sediment samples amplifiable by PCR using eubacterial 16S ribosomal DNA primers. The nahAc gene could also be amplified from indigenous bacteria by using nahAc-specific primers when PCR conditions were modified by increasing Taq and primer concentrations. Restriction digests of the nahAc amplification products from surface and subsurface sediments revealed polymorphism relative to P. putida G7. The procedures for DNA extraction, purification, and PCR amplification described here demonstrate that the PCR is a potentially useful tool in studies of function- and taxon-specific DNA from indigenous microbial communities in sediment and groundwater environments.
DOI：
10.1073/pnas.88.15.6667
发表时间：
1991-08-01
影响因子：
11.1
作者：
NELSON, K;WHITTAM, TS;SELANDER, RK
通讯作者：
SELANDER, RK
铜绿假单胞菌 lasA 基因的核苷酸序列和在大肠杆菌中的表达。
影响因子：
3.2
作者：
Schad,PA;Iglewski,BH
通讯作者：
Iglewski,BH