by Caitlin Smith
How long should it take to count molecules that you can’t see? Slowly and steadily, the process is getting faster, more efficient, and more accurate. Real-time PCR (or quantitative PCR, qPCR) is a method of quantifying the amount of DNA in a starting sample. In real-time PCR, DNA is amplified and detected in real time; this is distinct from standard PCR in which the amplified DNA is detected at the end of the reaction. Some methods use fluorescent dyes that intercalate with double-stranded DNA, while others use fluorescently-labeled oligonucleotide probes capable of emitting light after binding to their DNA target.
Now standard PCR is rivaled by “fast PCR,” in which a typical run can be done in under 30 minutes. This is accomplished by changes in instrumentation, enzymes, and protocols. Executing faster changes in temperature, using faster enzymes (without sacrificing accuracy), and shortening or even combining PCR steps all help to deliver faster PCR. These same “fast” principles are being applied to real-time PCR too – and with very promising results. If you use real-time PCR, take note: today’s “fast” real-time PCR is faster, more sensitive, and more accurate than the initial reports made it out to be.
Fast real-time PCR for the uninitiated
People are still learning about fast real-time PCR and what they can accomplish with it. “One of the most exciting new developments in this area today is the ability of fast qPCR to potentially bring qPCR technology to entirely new customers with unique challenges,” says Matthew Capizzi, associate product manager of PCR reagents in the molecular biology systems division at Life Technologies. “For example, for applications where turnaround time is paramount, waiting two hours for a result may be unacceptable, but attaining a result in under 40 minutes might open up a new window for qPCR.”
In fact, Capizzi believes that the main obstacle to advancing fast real-time PCR is not technological, but rather informational – many people simply don’t know enough about its advantages. “There is a significant percentage of the market that would greatly benefit from the time savings realized from the shorter runs produced by fast qPCR,” says Capizzi. “The challenge is educating and creating awareness for customers about this capability and having them try it out for themselves. Once they witness that the performance results they see are the same if not better than what they see in standard qPCR, they’ll never go back. It’s analogous to going from dial-up internet to broadband.” Life Technologies offers both instrumentation and reagents for fast real-time PCR. New systems include the StepOne™ and StepOnePlus™ Real-Time PCR Systems, and new reagents include the TaqMan® GTXpress™ Master Mix, Fast SYBR® Green Master Mix and Express qPCR SuperMix.
Learning to use fast real-time PCR has never been easier, even for fast PCR novices, in part because there are so many high quality reagent kits and systems available. Three examples among many are Qiagen, Applied Biosystems, and Kapa Biosystems. Qiagen’s offerings include their QuantiFast SYBR Green PCR Kit, QuantiFast Probe PCR Kit, and QuantiFast Multiplex PCR Kit. SABiosciences, a Qiagen company, offers their FAST SYBR® Green Master Mix, for shorter protocols, higher sensitivities, and sharper dissociation curves. Applied Biosystems’ 7900HT Fast Real-Time PCR System can handle up to 384-well plates, and has fully automated robotic loading. Finally, Kapa Biosystems offers their Kapa Probe Fast qPCR Kits that use fluorogenic probe chemistries such as hydrolysis, FRET, and displacement probes.
Even more data in less time
Sometimes you just need to crank through the experiments as quickly and efficiently as possible. Roche Applied Science’s LightCycler 1536 Real-Time PCR Instrument can run 1536 fast real-time PCR reactions in about 50 minutes. “The LightCycler 1536 Real-Time PCR Instrument is [an] exciting development because it gives researchers the ability to generate dramatically more data in a short amount of time,” says Mike Leous, group marketing manager for genomics and microarrays at Roche Applied Science. “Gene expression arrays will continue to have a role in discovery, but the speed of performing 1536 qPCR reactions in under an hour could create some occasions in which it makes more sense to run 1536 qPCR reactions than to run an array and follow it up with a 96-well or 384-well qPCR.”
The new LightCycler 1536 Real-Time PCR Instrument may help researchers overcome another challenge, according to Leous – the need for increased miniaturization. “People want to generate, for example, four times more data in less time – but they don’t have four times the money they had before,” he says. “Thus, with fast, high-throughput cycling, the challenge is miniaturizing without losing any quality of data or flexibility in experimental design. We believe the LightCycler 1536 Real-Time PCR Instrument, with its reaction volumes of 0.5-2 ml, can help researchers balance throughput and cost per data point – without sacrificing quality of data or experimental design.”
Improving and reinforcing fast amplification
Fast real-time PCR reagents require enzymes designed for this demanding job. Kamni Vijay, marketing manager for genomics at Bio-Rad Laboratories, says that “our mission is to develop amplification products with absolutely no compromise to sensitivity and dynamic range when moving from a standard to a fast qPCR format. We offer both the new line of SsoFast qPCR supermixes for real-time PCR, and the CFX96 and CFX384 real-time PCR detection systems. The SsoFast reagent products are based on our patented Sso7d fusion polymerase, allowing for greater sensitivity of detection and tolerance to inhibitors of PCR.” Indeed, Vijay says that recent fast real-time PCR is approaching the single-copy detection limits, even in complex samples such as whole blood or cell culture media.
Bio-Rad pairs amplification technology with CFX manager software for their detection systems, which is designed for a quick start regardless of the user’s experience level. “When we talk about fast real-time PCR, we don't just consider the actual run time,” says Vijay. “Instead, we consider time from reaction set-up to result.” Chemistry and instrumentation are advancing fast real-time PCR too. “[Advances] in fluorescent dyes are allowing more sensitive detection and better melt resolution capabilities, to achieve answers that had previously remained elusive,” she notes. “In instrumentation, we focus not only on how fast an instrument ramps, but rather how well a customer's reactions would work whenever they are performing fast PCR. We know that this requires not only faster ramp rates, but also exceptional temperature uniformity across the block, enabling shorter incubation times and drastically reduced overall run times. The newest fusion reagents we have developed accommodate these extreme ramping requirements and uniquely allow rapid amplification.”
Vijay echoes a sentiment similar to that of Life Technologies’ Capizzi: “Our biggest challenge today is in sharing with researchers that the technology has matured to a state that enables true fast real-time PCR. The perception that fast qPCR is somehow difficult to achieve or variable in its outcome is no longer the case. Thus, our efforts are focused around developing tools and applications that showcase refinements to the technology (in chemistry, hardware and software), and the simplicity with which fast results are now achievable.” Taking steps to show off the capabilities of today’s fast real-time PCR should prove fruitful – who wouldn’t want to save time in the lab?