PCR Gains Momentum with New Applications

From the archives of GEN, June 15, 2005


EDITOR’S NOTE: PCR has come a long, long way since it was first invented 36 years ago by the late Kary B. Mullis, as the article PCR Gains New Powers and Broadens Its Clinical Remit” by Vivienne Raper, PhD in this issue of GEN demonstrates. To give readers an even better sense of PCR’s evolution, we present below an excerpt from an article that appeared in GEN in 2005. The older article recalls a time when PCR was taking on an increasing variety of applications and making itself indispensible in many laboratories, especially drug discovery laboratories. The older article also highlights how PCR was responding to the “need for speed.”

Many companies in 2005 were already offering real-time PCR systems that could work with 96- and 384-well plates and robotic handlers. Another way to increase throughput was through multichannel detection, where four, five, or six different fluorescent dyes were used in the reaction. This enabled a researcher to combine this many reactions in a single vessel, then monitor the different reactions at different wavelengths.


PCR turns 20 this year, dating from its invention in 1985 by Kary B. Mullis. Since then, applications and adaptations of PCR have proliferated. The advent of real-time PCR couples a modified thermocycler with real-time kinetics, for a highly accurate method of quantitating the template DNA or RNA.

Additionally, manufacturers such as Applied Biosystems, Stratagene, and Ambion  have improved many other aspects of PCR, such as dyes and detection reagents, the speed of the procedure, and enzymes.

Real-time PCR differs from traditional, end-point PCR in one important aspect: The concentration of DNA is measured after each cycle, rather than at the end of the process only. The measurement is made through the use of fluorescent dyes.

When concentration is plotted against time, the result is a curve that is logarithmic in its early phases. This logarithmic plot is often referred to as “linear.” When concentrations are plotted on a log scale, the result is a straight line that can be used to calculate initial concentrations of template DNA with a high degree of accuracy.

This method is particularly attractive in the case of gene expression studies using mRNA. By looking at gene expression, researchers can detect changes due to a disease state or detect the presence of a pathogen, for example. Gene expression also provides clues about whether a drug compound is working or not.

Traditional methods for quantitation of mRNA such as northern blotting, in situ hybridization, and nuclease protection assays are falling out of favor due to inferior sensitivity and accuracy compared to real-time PCR.

The 7900HT system manufactured by Applied Biosystems is one of the more widely recognized real-time PCR instruments, and gene expression assays are one of its most popular applications.

Applied Biosystems product manager Chris Grimley reports, “Gene expression is our biggest application. We’re looking for changes in gene expression due to a disease state. A highly quantitative result is important.”

The 7900HT offers quantitative PCR with multiple channels for different fluorophores, gives results in about 35 minutes, and supports 96- or 384-well plate formats with robotic plate-handling capabilities.

A gene expression assay generally begins with mRNA, which is reverse transcribed to cDNA. The cDNA becomes the template for the PCR reaction. The demand for tools for RNA analysis, quantification, and amplification have exploded since Sayda Elbashir and Thomas Tuschle published their seminal paper on the subject in Nature in 2001 (24; 411(6836): 494–498).

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