Increasing Velocity of Reaction
There's also pure speed. Most lab workers are accustomed to waiting two hours or more for PCR reactions to finish. But several manufacturers are increasing the velocity of the reaction itself. Stratagene has abandoned taq polymerase entirely and has produced a DNA polymerase engineered from organisms of the kingdom Archea, which are frequently found in extreme environments and have proved to be a rich source of heat-tolerant enzymes.
These enzymes are marketed under the name FullVelocity. Stratagene says that with the FullVelocity enzymes, PCR can be completed in under an hour. However, Ann St. Louis, Stratagene's director of product marketing in gene expression, reports that in-house testing with specialized equipment resulted in total run times of 20 minutes.
"We have a high-speed amplification method that is another way to move the technology forward by increasing throughput," explains St. Louis. "There really is a need for faster instruments because the enzymes are faster than the instrument."
One example illustrating both the successful use of real-time PCR and an urgent need for faster, higher throughput reactions is a study authored by scientists from Komfo Anokye Teaching Hospital, Kumasi, Ghana (Transfusion 45(2):133). In the paper, Jean Pierre Allain and colleagues test a combined triple screen for HIV, hepatitis C, and hepatitis B for viral RNA and DNA in donated blood samples.
The triplex screen was found to be successful in identifying HIV and HCV RNA, but had a 1.55% false negative rate for HBV DNA. By pooling and testing blood ten samples at a time, costs can be kept low enough to screen all blood for infections, whereas under the previous system only some blood received adequate screening.
Another aspect of PCR speed is the physical limitation of the instrumentation. Whereas Stratagene cited mechanical limitations, Roche Applied Sciences (www.roche-applied-science.com) has optimized instrumental speed, but remains limited by its enzymes.
According to John Ogden, Ph.D., marketing manager at Roche Applied Sciences, in order to have good, accurate, sensitive results in PCR, it is important for the instrument to move as quickly as possible between temperature steps.
To that end, Roche designed the LightCycler, a system that controls the temperature of the reaction chamber with heated and cooled air. To maximize heat transfer, samples were contained in glass capillary tubes.
Until recently, this technology did not transfer to a block system. The temperatures of the blocks changed too slowly. However, Roche has solved the problem and is now releasing its new LightCycler 480.
"Now we can provide in a 96- or 384-well format everything people loved about LightCycler," says Dr. Ogden.
In response to the obvious synchronicity between Roche's LightCycler and Stratagene's FullVelocity enzymes, Dr. Ogden comments, "We haven't tried putting high-velocity enzymes with the LightCycler. It might be that somewhere in here there is this match of having fast enzymes and high-quality instrumentation and temperature control," but hastened to add that the LightCycler had only been validated with Roche's proprietary master mixes.
"If you mix and match between brands, it's difficult to know what you would get. I can't vouch or not vouch for that."
In the near future, scientists are looking forward to using rapid and quantitative PCR techniques to build miniature devices designed to give instantaneous results. QPCR may become the basis for a bedside molecular diagnostics tool or a handheld bioterror detection device. Many new applications are as-yet unconceived and will be inspired by the increasing power of the technique.