Loy Alexander, Pester Michael, Steger Doris
Department of Microbial Ecology, Faculty of Life Sciences, University of Vienna, Wien, Austria.
Methods Mol Biol. 2011;688:187-206. doi: 10.1007/978-1-60761-947-5_13.
High-throughput sequencing and hybridization technologies promise new insights into the natural diversity and dynamics of microorganisms. Among these new technologies are phylogenetic oligonucleotide microarrays (phylochips) that depend on the standard molecules for taxonomic and environmental studies of microorganisms: the ribosomal RNAs and their encoding genes. The beauty of phylochip hybridization is that a sample can be analyzed with hundreds to thousands of rRNA (gene)-targeted probes simultaneously, lending itself to the efficient diagnosis of many target organisms in many samples. An emerging application of phylochips is the highly parallel analysis of structure-function relationships of microbial community members by employing in vivo substrate-mediated isotope labeling of rRNA (via the isotope array approach). This chapter provides an introduction to phylochip and isotope array analysis and detailed wet-lab protocols for preparation, labeling, and hybridization of target nucleic acids.
高通量测序和杂交技术有望为微生物的自然多样性和动态变化带来新的见解。这些新技术中包括系统发育寡核苷酸微阵列（系统发育芯片），其依赖于用于微生物分类学和环境研究的标准分子：核糖体RNA及其编码基因。系统发育芯片杂交的优点在于，一个样本可以同时用数百至数千个靶向rRNA（基因）的探针进行分析，从而能够高效诊断多个样本中的多种目标生物体。系统发育芯片的一个新兴应用是通过对rRNA进行体内底物介导的同位素标记（通过同位素阵列方法），对微生物群落成员的结构-功能关系进行高度并行分析。本章介绍了系统发育芯片和同位素阵列分析，并提供了用于靶核酸制备、标记和杂交的详细湿实验方案。
