Quantitative real-time PCR for studies of microbial gene expression

现代微生物学，像许多生命科学一样，需要应用分子方法来解决基因多样性，丰度和调控问题。聚合酶链反应（PCR）是分子微生物学工具箱的日常主食，因为它允许扩增单个基因靶，用于序列分析、克隆、核酸杂交和许多其他应用。在过去的几年中，仪器已经变得容易用于实时聚合酶链式反应，其中可以测量PCR反应期间单基因扩增的速率，以定量样品中特定模板分子的原始数量。定量实时PCR（qPCR）正迅速成为一种不可或缺的技术，甚至正在取代用于定量基因表达（一旦RNA分子转化为cDNA）的北方印迹杂交等分子生物学经典技术。我的工作包括比较全球基因表达（通过cDNA阵列杂交）在高和低氧压力下，并在暴露于亚硝化剂，亚硝酸盐和一氧化氮的氨氧化细菌的多个物种，找到基因参与亚硝化应激反应和一氧化二氮的生产。一旦基因被识别，它们在野生型和突变背景下的调节和生理功能就会被仔细检查，并且相同的基因被用来检查它们在自然生态系统样品中的表达水平。所有这些活动都需要qPCR：1）验证来自微阵列杂交的全基因组表达数据，2）确定突变与野生型细菌或暴露于不同刺激（例如亚硝酸盐，低氧，一氧化氮）的细菌中基因表达的相对水平，以及3）确定从环境样品中提取的DNA或RNA混合物中基因模板的原始量。qPCR是非常强大的，因为它的检测限低，能够扩增模板的任何部分，不仅允许查询基因的直接表达，而且还允许查询RNA的加工，以及通过使用多个荧光团在单个PCR反应中检测多个模板的能力。

Modern day microbiology, like many life sciences, necessitates the application of molecular methods to address questions of gene diversity, abundance, and regulation. The polymerase chain reaction (PCR) is an everyday staple of the molecular microbiology tool chest as it allows for the amplification of individual gene targets for sequence analysis, cloning, nucleic acid hybridization, and many other applications. In the past few years, instrumentation has become readily available for real-time polymerase chain reaction, wherein one can measure the rate of single gene amplification during the PCR reaction in order to quantify the original number of a specific template molecule in a sample. Quantitative real-time PCR (qPCR) is rapidly becoming an indispensable technique and is even replacing such molecular biology classics as Northern blot hybridization for quantifying gene expression (once RNA molecules are converted into cDNA). My work involves comparing global gene expression (via cDNA array hybridization) in multiple species of ammonia-oxidizing bacteria under high and low oxygen tension and upon exposure to the nitrosating agents, nitrite and nitric oxide, to find genes involved in nitrosative stress response and nitrous oxide production. Once the genes are identified, their regulation and physiological function in wild-type and mutant backgrounds are carefully examined, and the same genes are used to examine levels of their expression in samples from natural ecosystems. All of these activities require qPCR to: 1) verify whole-genome expression data derived from microarray hybridization, 2) determine relative levels of gene expression in mutated versus wild-type bacteria, or bacteria exposed to different stimuli (e.g. nitrite, low oxygen, nitric oxide), and 3) determine the original amount of a gene template in a mixture of DNA or RNA extracted from environmental samples. qPCR is extremely powerful due to its low detection limit, the ability to amplify any part of a template, allowing one to query not just direct expression of a gene, but also the processing of RNA, and the ability to detect multiple templates in a single PCR reaction by using multiple fluorophores.