直接基因荧光原位杂交（geneFISH）技术：在单细胞水平上连接微生物与其基因。

Linking Microbes to Their Genes at Single Cell Level with Direct-geneFISH.

机构信息

Department of Environmental Technology, Technische Universität Berlin, Berlin, Germany.

Institute for Chemistry and Biology of the Marine Environment, Oldenburg, Germany.

出版信息

Methods Mol Biol. 2021;2246:169-205. doi: 10.1007/978-1-0716-1115-9_12.

DOI:10.1007/978-1-0716-1115-9_12

PMID:33576990

Abstract

Direct-geneFISH is a Fluorescence In Situ Hybridization (FISH) method that directly links gene presence, and thus potential metabolic capabilities, to cell identity. The method uses rRNA-targeting oligonucleotide probes to identify cells and dsDNA polynucleotide probes carrying multiple molecules of the same fluorochrome to detect genes. In addition, direct-geneFISH allows quantification of the cell fraction carrying the targeted gene and the number of target genes per cell. It can be applied to laboratory cultures, for example, enrichments and phage infections, and to environmental samples. This book chapter describes the main steps of the direct-geneFISH protocol: probe design and synthesis, the "core" direct-geneFISH protocol and lastly, microscopy and data analysis.

摘要

直接基因 FISH 是一种荧光原位杂交（FISH）方法，它将基因的存在，以及潜在的代谢能力，直接与细胞身份联系起来。该方法使用 rRNA 靶向寡核苷酸探针来识别细胞，并用携带相同荧光染料的多个分子的 dsDNA 多核苷酸探针来检测基因。此外，直接基因 FISH 允许定量携带靶向基因的细胞分数和每个细胞的目标基因数量。它可以应用于实验室培养物，例如富集物和噬菌体感染，以及环境样本。本章描述了直接基因 FISH 方案的主要步骤：探针设计和合成、“核心”直接基因 FISH 方案以及最后是显微镜和数据分析。