Carl Wittwer, the University of Utah scientist who helped invent 15-minute PCR and many other commonly used PCR techniques and instrumentation, is back at the drawing board, whittling the time it takes to perform PCR to less than 30 seconds.
By employing high surface area-to-volume capillary tubes and up to 25-fold higher primer and polymerase concentrations than standard PCR, Wittwer and colleagues have used the method to successfully amplify a 49-bp fragment of human genomic DNA in less than 30 seconds and 102-bp fragments in less than a minute.
Although the technique is in the very early proof-of-principle stage, using relatively crude laboratory equipment such as water baths and a stepper motor, it demonstrates the potential of PCR amplification as a true point-of-care molecular testing tool.
In addition, PCR Insider has learned that Wittwer's lab has collaborated with Canon US Life Sciences to successfully perform the PCR method along with melt curve analysis in less than three minutes on Canon's prototype microfluidics-based genotyping platform.
Wittwer, a professor of pathology at the University of Utah Medical School, and medical director of the Advanced Technology Group at ARUP Laboratories, is widely credited with having developed the rapid-cycle (10- to 15-minutes) PCR method that serves as the basis of Roche's well-known LightCycler PCR products.
He also adapted flow cytometry optics to thermal cycling to enable real-time monitoring of PCR; introduced SYBR Green I, fluorescent hybridization probes, and melting analysis to real-time PCR; and co-founded BioFire Diagnostics (previously known as Idaho Technology), which currently sells instrumentation and reagents for real-time PCR and high-resolution melt analysis for clinical diagnostics, biodefense, and other applications.
In October at the Association for Molecular Pathology meeting in Long Beach, Wittwer and Jared Farrar, a laboratory specialist in the Department of Pathology at the University of Utah School of Medicine, presented a poster detailing the so-called "extreme" less-than-30-second PCR technique. Wittwer provided an update on his group's work on the method last week at Cambridge Healthcare Institute's inaugural Integrating Digital PCR conference in Boston.
Wittwer noted during his presentation that about 2.6 seconds per cycle had recently been established in the literature as the threshold for completing significant PCR amplification, which, if done for a typical 35 cycles or so, would equate to about 1.5 minutes for meaningful target amplification.
PCR Insider reported in 2011 on a group led by Reginald Beer at Lawrence Livermore National Laboratory that pushed PCR to the sub-three-minute range using microliter-scale reaction volumes (PCR Insider, 8/4/2011).
However, most previously published methods have generally amplified less-complex targets such as plasmids, PCR products, viruses, and bacteria, at extremely high initial template concentrations, often leading to decreased PCR efficiency or failure at faster cycle times.
The technique employed by Wittwer and Farrar, compared with 12-minute rapid PCR, uses a 25-fold greater concentration of polymerase and primers. This modification has been attempted by others in rapid PCR, Wittwer said, but generally produces lots of "junk" products.
Recognizing that thermal cycling speed in a PCR reaction is largely governed by the characteristics of the sample container, the Wittwer lab attempted to combine these high polymerase and primer concentrations with glass or stainless steel capillary tubes featuring a high surface area-to-volume ratio.
To conduct PCR amplification, they inserted a micro-thermocouple inside the reaction tubes for accurate temperature measurement and achieved temperature cycling using a stepper motor to quickly rotate samples between two stirred water baths held at constant temperatures, for instance, 95°C and 40°C — very similar to the way PCR was done for several years after its invention.
While the technique "could work outside of water baths — maybe using microfluidics — getting those temperature changes is very hard to do," Wittwer told PCR Insider following his presentation. "We used water baths only because it was the easiest way to change the temperature rapidly and reproducibly."
Using their method, the researchers were able to achieve PCR with cycle times of less than two seconds, and in some cases as low as 0.5 seconds per cycle. More specifically, they were able to consistently amplify a 45-bp fragment of KCNE1 and a 49-bp fragment of IRL10RB from human genomic DNA in less than 30 seconds, at 0.8 seconds per cycle; and a 102-bp fragment of NQO1 in less than one minute, at 1.93 seconds per cycle.
Wittwer said that the method still has to overcome several significant challenges and concerns before it could be practically implemented. The higher primer concentration is an issue, but not as much as it used to be since "primers are cheap" nowadays, Wittwer said.
Of more concern is the potential high cost of using 20-fold or greater concentrations of polymerase, he noted. So far the Wittwer lab has successfully used KlenTaq polymerase, a version of Taq polymerase that is sold by several companies and has recently become more affordable due to the expiration of patent protection. Wittwer noted that Boston-based Enzymatics has thus far offered the best price on KlenTaq, but that using high concentrations of the enzyme is still expensive.
He also said that the method may work even better with KAPA2G fast DNA polymerase from Kapa Biosystems. Engineered for higher processivity and speed, and offering significantly faster extension rates and improved PCR success rates than wild-type Taq polymerase, KAPA2G was also mentioned by LLNL's Beer as one of only two enzymes that worked with their sub-three-minute PCR technique. However, using this enzyme at the necessary high concentrations may also be prohibitively expensive.
More importantly, Wittwer noted, is the lack of instrumentation available to perform the 30-second-or-less PCR method. "The limitation of PCR is not the biochemistry," he said. "It's still the instrumentation."
On that point, however, Wittwer said that his lab has been working with Canon US Life Sciences to "try and marry extreme PCR with melt curve analysis" on Canon's microfluidics-based genotyping platform, which is still under development at the company.
Based on microfluidics IP licensed from Caliper Life Sciences (now PerkinElmer), as well as consumer electronics-inspired detection technology developed in house, the Canon platform has been under development for several years and is expected to be an automated, cartridge-based system for rapid molecular testing and genotyping.
Canon has not released many details on the platform's development over the past several months, but Wittwer said that the prototype that his lab was using could test eight patient DNA samples in parallel in discrete 50-µL volumes. "It's the closest instrument to being able to do [the sub-30-second PCR method] outside of water baths," Wittwer said.
In an email to PCR Insider, Renee Howell, director of R&D at Canon US Life Sciences, said that under a research partnership with Canon, the Wittwer lab has "a prototype instrument designed and built by Canon US Life Sciences that is allowing them to explore ultra-fast PCR chemistry;" and that the instrument "uses a microfluidic chip with embedded heaters that cycles temperature fast enough to meet" the requirements of the technique.
"Recently, [the Wittwer] lab also successfully completed an independent trial of the prototype instrument's capability to perform genotyping on clinical samples using PCR and HRM," Howell said. "Canon US Life Sciences is planning on having an early access version of the instrument for research work available next year and is currently looking for candidate labs that would participate in the early access program."
Wittwer said that early data from his lab's work with the platform suggest that it can complete the ultra-fast PCR in one to two minutes, as well as subsequent HRM analysis in about one minute.
Perhaps the most important question surrounding the new PCR method is, "Why would anyone want to do it so quickly?" Wittwer queried.
He noted that when he first developed the 15-minute PCR technology destined to serve as the basis of the Roche LightCycler, ARUP Labs provided significant assistance in implementing the technology for clinical use.
"However, 30-second PCR in a reference lab … where they collect 100 samples and do them all at once … is not of great interest" Wittwer said. However, for near-patient diagnostics — or even home testing — the technique could have tremendous potential, he added.