Optimizations of rRNA-based Microbial Analyses

描述（由申请人提供）：核糖体RNA（rRNA）基因分析是表征环境中存在的生物体（包括与医疗状况相关的生物体）的最重要方法之一。这些分析的第一步通常是应用聚合酶链反应（PCR）来特异性扩增感兴趣的基因。这需要使用对rRNA基因具有特异性的引物，但能够容纳基因的所有天然变异。研究人员将分析目前可用数据中rRNA基因引物结合位点的序列变异，目的是改进目前的引物设计，以识别所有相关生物体。他们还将探索使用序列简并性，非规范核苷酸和引物退火严格性以适应观察到的序列多样性的权衡。在这样做的过程中，他们将探索可能引导扩增朝向共同模板和引物序列的策略的有用性。这些目标将使用计算机分析数据库中的序列，并对替代引物设计和扩增方案进行实验比较。大部分工作的结果将适用于基于rRNA基因以外的分子的分子分析。微生物在人类健康和疾病中发挥着重要作用。有越来越多的研究与正常和异常条件相关的微生物的混合物。最深入的研究是基于对特定分子的分析，最常见的是核糖体RNA。拟议研究的目标是改进这些方法，以检测样品中存在的生物体的全部多样性。这项工作将与正在进行的人类阴道微生物群分析相结合。

DESCRIPTION (provided by applicant): Analysis of ribosomal RNA (rRNA) genes is one of the most important methods for characterizing the organisms present in an environment, including those associated with medical conditions. The first step of these analyses is usually to apply the polymerase chain reaction (PCR) to specifically amplify the genes of interest. This requires the use of primers that are specific to the rRNA genes, yet capable of accommodating all of the natural variation of the genes. The investigators will analyze sequence variation in rRNA gene primer-binding sites in the currently available data with the goal of improving current primer designs to recognize the full range of relevant organisms. They will also explore trade-offs in using sequence degeneracy, noncanonical nucleotides, and primer annealing stringency to accommodate the observed sequence diversity. In so doing, they will explore the usefulness of strategies that might guide amplifications toward a common template and primer sequence. These goals will use computer analysis of sequences from the databases and experimental comparisons of alternative primer designs and amplification protocols. The results of much of the proposed work will be applicable for molecular analyses based on molecules other than rRNA genes. PUBLIC HEALTH RELEVANCE Microbes play important roles in human health and disease. There are increasing numbers of studies of the mixture of microbes associated with both normal and abnormal conditions. Most in-depth studies are based upon the analysis of particular molecules, most commonly the ribosomal RNAs. The goal of the proposed studies is to improve these methods in the detection of the full diversity of organisms present in the samples. This work will be integrated with on-going analyses of vaginal microbiota in humans.