Development of a two-dimensional electrophoresis method to study soil bacterial diversity
Development of a two-dimensional electrophoresis method to study soil bacterial diversity
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DOI：
10.1111/j.1747-0765.2006.00081.x
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发表时间：
2006-10
影响因子：
2
通讯作者：
Takumi Isshi;Rajendran Narasimmalu;T. Amemiya;K. Itoh
中科院分区：
文献类型：
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作者：
Takumi Isshi;Rajendran Narasimmalu;T. Amemiya;K. Itoh
摘要近年来，以聚合酶链反应（PCR）为基础的非培养方法已发展用于环境细菌的研究。变性梯度凝胶电泳（DGGE）是这些方法中最有用的一种。然而，在DGGE方法中，与土壤细菌群落的高多样性相比，凝胶电泳的低分辨率可能是一个问题。土壤群落含有数千种细菌，但在凝胶上分辨超过20-50个条带是困难的。为了克服这些问题，我们设计了一种结合大小分离和DGGE方法的二维电泳（2-DE）方法。在本研究中，首次使用细菌DNA设计了一种使用细菌16 S rDNA的PCR产物的2-DE方案。通过比较不同的2-DE方法，本研究中描述的2-DE可以成功地分离细菌16 S rDNA。该方案应用于环境样品（即土壤），结果表明，一个带的大小分离或DGGE产生两个或两个以上的点后，2-DE，从而提供额外的有用信息的细菌种群在环境中。我们的研究结果表明，新的2-DE方法可能是有用的监测土壤微生物种群的细节。
Abstract In recent years, culture-independent methods based on polymerase chain reaction (PCR) have been developed for the study of environmental bacteria. Denaturing gradient gel electrophoresis (DGGE) is one of the most useful of these methods. However, in the DGGE method, the low resolution of gel electrophoresis compared with the high diversity of soil bacterial communities can be a problem. Soil communities contain several thousand species of bacteria, but the resolution of more than 20–50 bands on a gel is difficult. To overcome these problems, we designed a two-dimensional electrophoresis (2-DE) method that combines size separation and the DGGE methods. In the present study, bacterial DNA was first used to design a protocol for 2-DE using PCR products of bacterial 16S rDNA. After examining different 2-DE approaches, the bacterial 16S rDNA could be separated successfully using the 2-DE described in this study. This protocol was applied to an environmental sample (i.e. soil) and the results showed that one band of size separation or DGGE produced two or more spots after 2-DE, thereby providing additional useful information about the bacterial population in the environment. Our results suggest that the new 2-DE method may be useful to monitor soil microbial populations in detail.