Phadke Sujal, Salvador Andreia Filipa, Alves Joana Isabel, Bretschger Orianna, Alves Maria Madalena, Pereira Maria Alcina
J. Craig Venter Institute, 4120 Capricorn Lane, La Jolla, CA, 92037, USA.
CEB-Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal.
Methods Mol Biol. 2017;1620:225-248. doi: 10.1007/978-1-4939-7060-5_16.
Polymerase chain reaction (PCR) is central to methods in molecular ecology. Here, we describe PCR-dependent approaches useful for investigating microbial diversity and its function in various natural, human-associated, and built environment ecosystems. Protocols routinely used for DNA extraction, purification, cloning, and sequencing are included along with various resources for the statistical analysis following gel electrophoresis-based methods (DGGE) and sequencing. We also provide insights into eukaryotic microbiome analysis, sample preservation techniques, PCR troubleshooting, DNA quantification methods, and commonly used ordination techniques.
聚合酶链反应(PCR)是分子生态学方法的核心。在此,我们描述了依赖PCR的方法,这些方法对于研究各种自然、人类相关和人造环境生态系统中的微生物多样性及其功能很有用。文中包括了常规用于DNA提取、纯化、克隆和测序的方案,以及基于凝胶电泳方法(DGGE)和测序后的统计分析的各种资源。我们还提供了关于真核生物微生物组分析、样品保存技术、PCR故障排除、DNA定量方法和常用排序技术的见解。