Real Time PCR

Quantitative PCR with no gel? Impossible! Now with the ABI PRISM 7700 Sequence Detection System, real time PCR has become a reality. Combining a novel chemistry with a fully integrated PCR amplification and detection system, the 7700 System is a unique, closed-tube system that allows for real time quantitative PCR without sample processing or electrophoresis. At the core of the system is a 5' nuclease assay conducted with the TaqMan¨ PCR Reagent Kit utilizing a fluorogenic probe that has b

Nov 24, 1997
The Scientist Staff

Quantitative PCR with no gel? Impossible! Now with the ABI PRISM 7700 Sequence Detection System, real time PCR has become a reality. Combining a novel chemistry with a fully integrated PCR amplification and detection system, the 7700 System is a unique, closed-tube system that allows for real time quantitative PCR without sample processing or electrophoresis.

At the core of the system is a 5' nuclease assay conducted with the TaqMan¨ PCR Reagent Kit utilizing a fluorogenic probe that has both a reporter and quencher dye attached. When both dyes are attached to the probe, reporter dye emission is quenched. However, once annealed to the target sequence and put into a PCR reaction, the nuclease activity of the Taq polymerase cleaves the reporter dye from the probe, resulting in a fluorescent signal. The fluorescent signal is monitored at every cycle as additional reporter dye molecules accumulate.

Real time PCR offers numerous advantages over previous attempts at quantitating PCR. Other methods typically rely on end-point measurements, when often the reaction has gone beyond the exponential phase because of limiting reagents. To compensate for such problems, competitive PCR was devised, which allows for normalization of the end product based on the ratio between target and competitor. Because this method is cumbersome, requiring a carefully constructed competitor target for each PCR reaction and a series of dilutions to ensure that there is a suitable ratio of target to competitor, it is seldom used successfully. In contrast, with real time PCR, the dynamic range is much greater than that of competitive PCR (over six orders of magnitude as compared to one with competitive PCR), post-reaction processing is eliminated, and the measurements are taken from the exponential range of the reaction, where component concentrations are not limiting. And best of all, the entire process is automated.

The ABI 7700 Sequence Detection System includes a built-in thermal cycler, a fluorogenic 5' nuclease assay, a laser for inducing fluorescence, charge-coupled device (CCD) detection, and PCR application software. The specially designed reaction tube with transparent lid allows light from a laser, carried on fiber optic cables, to excite the probe and allows the emitted light to be sent back to a CCD camera for detection without the user ever having to open the tube. Being a closed tube system all the way through to the detection phase eliminates the possibility of contamination and the necessity for special procedures and rooms. Designed for high- throughput applications, the 7700 System collects fluorescent emission between 500 and 660 nm in each of 96 sample wells every few seconds and displays results in one minute after the reaction is completed.

While not inexpensive, costing in the vicinity of $95,000, to some users, the savings in labor and in the expense of processing samples has actually made PCR more affordable. Of the ABI PRISM 7700, Mickey Williams of Genetech said, "In the past, PCR was simply too costly and time-consuming to be used as a routine test . . ." "A project that used to take weeks can now be completed in a matter of days." Charlotte Ip, Senior Research Fellow at Merck, said of the 7700 System,"After using other types of quantitative PCR methods, this system is by far the best. It has many applications and performs very well [both with respect to] quantitation and accuracy."

The only one of its kind in the market, the PRISM 7700 system is able to analyze up to 500 samples a day, vastly more than any other kind of PCR analysis. With this system, time required to screen drugs for effects on gene expression should be significantly accelerated. A single computer with a graphical user interface controls not only data analysis but also set-up of PCR conditions, allowing fast and convenient programming of PCR reactions.

For more information, visit Perkin-Elmer's web site at www.perkin-elmer.com, or call the company sales office at 800-345-5224.