Real-Time PCR Approaches for Analysis of Hydrocarbon-Degrading Bacterial Communities

Abstract

Since the development of the polymerase chain reaction (PCR) in the 1980s, our knowledge of environmental microbial diversity and function has increased greatly. However, quantification of particular environmental microbes by “endpoint PCR” techniques has typically been inaccurate due to inherent limitations and biases introduced during amplification. Such problems were overcome in the 1990s following the development of “real-time PCR” methods that employ highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction as each cycle occurs (i.e. in real time). Real-time PCR is now widely employed for measuring 16S rRNA gene and functional gene abundance and expression in the environment and has included numerous studies of hydrocarbon-degrading bacteria, and the technique has promising possibilities as a tool for assessing hydrocarbon-contaminated environments and monitoring natural attenuation or bioremediation techniques. This chapter looks at the kinetics of PCR to explain the benefits of real-time PCR over traditional endpoint PCR and discusses the most popular detection chemistries and how they allow accurate quantification. Guidelines are provided for the design of real-time PCR primers and probes, and detailed protocols are given for both TaqMan and SYBR Green assays for quantifying gene abundance, as well as a two-step reverse transcription real-time PCR protocol for quantifying gene expression.