Department of Civil and Environmental Engineering, National University of Singapore, Singapore 117576.
Environ Microbiol. 2011 Sep;13(9):2565-75. doi: 10.1111/j.1462-2920.2011.02527.x. Epub 2011 Aug 8.
Fingerprinting techniques provide access to understanding the ecology of uncultured microbial consortia. However, the application of current techniques such as terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) has been hindered due to their limitations in characterizing complex microbial communities. This is due to that different populations possibly share the same terminal restriction fragments (T-RFs) and DNA fragments may co-migrate on DGGE gels. To overcome these limitations, a new approach was developed to separate terminal restriction fragments (T-RFs) of 16S rRNA genes on a two-dimensional gel (T-RFs-2D). T-RFs-2D involves restriction digestion of terminal fluorescence-labelled PCR amplified 16S rRNA gene products and their high-resolution separation via a two-dimensional (2D) gel electrophoresis based on the T-RF fragment size (1(st) D) and its sequence composition on the denaturing gradient gel (2(nd) D). The sequence information of interested T-RFs on 2D gels can be obtained through serial poly(A) tailing reaction, PCR amplification and subsequent DNA sequencing. By employing the T-RFs-2D method, bacteria with MspI digested T-RF size of 436 (±1) bp and 514 (±1) bp were identified to be a Lysobacter sp. and a Dehalococcoides sp. in a polychlorinated biphenyl (PCB) dechlorinating culture. With the high resolution of 2D separation, T-RFs-2D separated 63 DNA fragments in a complex river-sediment microbial community, while traditional DGGE detected only 41 DNA fragments in the same sample. In all, T-RFs-2D has its advantage in obtaining sequence information of interested T-RFs and also in characterization of complex microbial communities.
指纹图谱技术可用于了解未培养微生物群落的生态。然而，由于其在表征复杂微生物群落方面的局限性，目前的技术（如末端限制性片段长度多态性（T-RFLP）和变性梯度凝胶电泳（DGGE））的应用受到了阻碍。这是因为不同的种群可能具有相同的末端限制性片段（T-RFs），并且 DNA 片段可能在 DGGE 凝胶上共迁移。为了克服这些限制，开发了一种新方法来分离 16S rRNA 基因的末端限制性片段（T-RFs）在二维凝胶（T-RFs-2D）上。T-RFs-2D 涉及末端荧光标记的 PCR 扩增 16S rRNA 基因产物的限制性消化，以及通过基于 T-RF 片段大小（1（st）D）和其在变性梯度凝胶（2（nd）D）上的序列组成的二维（2D）凝胶电泳进行高分辨率分离。通过连续的 poly(A) 加尾反应、PCR 扩增和随后的 DNA 测序，可以获得感兴趣的 T-RFs 在 2D 凝胶上的序列信息。通过使用 T-RFs-2D 方法，从多氯联苯（PCB）脱氯培养物中鉴定出 MspI 消化的 T-RF 大小为 436（±1）bp 和 514（±1）bp 的细菌为 Lysobacter sp. 和 Dehalococcoides sp.。通过 2D 分离的高分辨率，T-RFs-2D 分离了复杂河流沉积物微生物群落中的 63 个 DNA 片段，而传统的 DGGE 在相同的样品中仅检测到 41 个 DNA 片段。总之，T-RFs-2D 具有获取感兴趣的 T-RFs 的序列信息的优势，并且还具有表征复杂微生物群落的优势。
