Marni Brisson, Larissa Tan, Rob Park, and Keith Hamby, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Dr., Hercules, CA 94547 USA
Real-time PCR* is a powerful and effective technique for accurate quantitation of DNA. Assays for the detection of a single gene involve careful choice of primers, target sequence, and the method for detecting the amplified DNA product. In addition, it is necessary to choose appropriate reaction conditions that will generate robust and efficient amplification for both relative and absolute quantitation.
Specificity and efficiency are two essential requirements for successful real-time PCR amplification. Poor assay design can result in such problems as nonspecific products and inefficient primer association with target, which dramatically affect the quality of the data. Proper primer design ensures that the chosen primers are specific for the desired target sequence, bind at positions that avoid secondary structure, and minimize the occurrence of primer-dimer formation. Appropriate reaction conditions can also improve the efficiency of amplification. Optimization of the detection method, Mg2+ concentration, annealing temperature, enzyme concentration, PCR product length, etc., are all ways of perfecting real-time assays.
Another valuable tool in assay design is melt-curve analysis. With the
use of DNA-binding dyes such as SYBR Green I, a melt-curve profile can
be generated. The iCycler iQ system records the total fluorescence generated
by SYBR Green I binding to double-stranded DNA as temperature changes,
and plots the fluorescence in real time as a function of temperature.
The first derivative of this plot, dF/dT, is the