Faye Boeckman, Marni Brisson, and Larissa Tan, Bio-Rad Laboratories, Inc., Hercules CA 94547 USA
The polymerase chain reaction (PCR) has proven to be a versatile tool in molecular biology. The use of this technique has generated unprecedented advances in gene discovery, diagnostics, and gene expression analysis. In addition, new techniques that build on PCR have further expanded its range of scientific applications.
Real-time PCR is a powerful advancement of the basic PCR technique. Through the use of appropriate fluorescent detection strategies in conjunction with proper instrumentation, the starting amount of nucleic acid in the reaction can be quantitated. Quantitation is achieved by measuring the increase in fluorescence during the exponential phase of PCR. Applications of real-time PCR include measurements of viral load, gene expression studies, clinical diagnostics, and pathogen detection.
Although the performance of PCR in more routine molecular biology applications can be relatively straightforward to optimize, several parameters must be evaluated and optimized independently to achieve the maximum potential of real-time PCR. The factors that affect real-time PCR fall into 3 categories. These are general laboratory practices, template and primer design, and reaction components and conditions. When determining which conditions to optimize, the ultimate assay goal (i.e., qualitative analysis vs. quantitation) must be considered.
To develop sensitive real-time PCR assays cost effectively, you should
design and optimize the primer sets prior to developing the probe. This
technical note will guide you in the development of an opti