Zwirglmaier Katrin, Fichtl Katrin, Ludwig Wolfgang
Lehrstuhl für Mikrobiologie, Technical University Munich, Freising, Germany.
Methods Enzymol. 2005;397:338-51. doi: 10.1016/S0076-6879(05)97020-1.
Fluorescence in situ hybridization (FISH) using specific probes certainly is one of the most commonly applied molecular techniques in microbial ecology. Monitoring of community composition and dynamics can be combined with localization and identification of individual cells in situ. However, the resolution power of the method is limited by the need for high target numbers per cell. Apart from standard targets (ribosomal RNAs), mRNAs could be used successfully for in situ visualization in some cases. A new promising variant of in situ hybridization could be established that should provide access to any low copy number nucleic acid targets, such as chromosomal genes. The recognition of individual genes by FISH (RING-FISH) technology is based on target visualization mediated by polynucleotide probe network formation. The specificity of the approach is provided by intracellular probe-target hybridization. This initial hybridization apparently acts as a focal point for inter-probe hybridization within and mainly in the periphery of the cells. Probe-conferred fluorescence typically appears halo-like around the cells. RING-FISH can be used in combination with conventional oligonucleotide FISH. Thus, genetic potential can be assigned to in situ identified cells.
使用特异性探针的荧光原位杂交（FISH）无疑是微生物生态学中最常用的分子技术之一。群落组成和动态监测可与原位单个细胞的定位和鉴定相结合。然而，该方法的分辨率受到每个细胞所需高靶标数量的限制。除了标准靶标（核糖体RNA）外，在某些情况下，mRNA也可成功用于原位可视化。可以建立一种新的有前景的原位杂交变体，该变体应能够检测任何低拷贝数的核酸靶标，如染色体基因。通过FISH（RING-FISH）技术识别单个基因是基于由多核苷酸探针网络形成介导的靶标可视化。该方法的特异性由细胞内探针-靶标杂交提供。这种初始杂交显然作为细胞内和主要在细胞周边的探针间杂交的焦点。探针赋予的荧光通常在细胞周围呈晕圈状出现。RING-FISH可与传统的寡核苷酸FISH结合使用。因此，可以将遗传潜力赋予原位鉴定的细胞。
