Technology Corner
Extreme PCR: A Breakthrough Innovation for Outbreaks? Vikram Sheel Kumar1* and Molly Webster2
The latest Ebola outbreak is the diagnostics grand challenge of the moment. Groups across the world are racing to build the “ideal test” that according to the WHO should work without laboratory infrastructure, take fewer than 3 steps, report results in under 30 min, and have minimum biosafety requirements (1 ). Leo Poon, head of Public Health Laboratory Sciences at the University of Hong Kong School of Public Health, who was one of the first to decode the SARS (severe acute respiratory syndrome) virus and develop a test for H1N1 influenza, would add “affordable” to that list. “What we need in the field is something simple; and it must be cheap,” he says. What is the most radical innovation with the potential to deal with viral outbreaks? “The portable PCR machine,” Poon shares. Seven thousand miles away a pathologist in Utah may have hit on an innovation that will change PCR forever. Dr. Carl Wittwer, professor of pathology at the University of Utah Medical School, has been recognized by the inventor of PCR, Kary Mullis, as “someone who has thought about PCR in a way that very few others have” (2 ). Wittwer invented the LightCycler system, which was licensed to Roche with over 10 000 units placed across the world. He spoke with this journal about his latest innovation, “extreme PCR.” Why Is This Innovation Important?
Carl Wittwer
“Once a technology becomes established, further innovation is still possible.” says Wittwer, referring to PCR. The dependence on central laboratory testing constrains the ability for a community to deal with an outbreak such as Ebola. The motivation for decentralization is to obtain faster results to provide medical action
1 Boston Children’s Hospital, Boston, MA; 2 Science writer and producer, Brooklyn, NY. * Address correspondence to this author at: Boston Children’s Hospital, 390 Commonwealth Ave., Apt. 605, Boston, MA 02215. Fax 617-899-8944; e-mail [email protected]. Received January 14, 2015; accepted January 16, 2015. DOI: 10.1373/clinchem.2014.236950 © 2015 American Association for Clinical Chemistry
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Clinical Chemistry 61:4 (2015)
in the field. “That is a place where historically PCR has not been very competitive because of the time required just to amplify—an hour or two. When you show you can amplify in less than a minute, now you can do nucleic acid testing in a very short time,” explains Wittwer, whose rapid-cycle PCR technology that brought PCR time down to 10 –30 min dates back to the 1990s (3 ). “A number of advances including microfluidics showed that PCR in less than 10 minutes was feasible, but the quality of amplification became poor when limited with time,” says Wittwer. “We changed the chemistry of the reaction.” How Does It Work? “This is pretty obvious, and I kick myself for not doing it 20 years ago. The information was all there,” he shares. PCR has 3 steps: annealing, extension, and denaturation. The “critical thing” in PCR is getting the solutions to the required temperatures. Conventional instruments do this poorly, and have to Leo Poon add additional holding times in order for the sample to reach temperature targets. But according to Wittwer, a trick was needed in chemistry, not instrumentation (Fig. 1). Annealing occurs between primers and singlestranded DNA targets. Because there are 2 oligonucleotides binding, it is a “second-order issue.” However, “for most of PCR you have millions of copies of primers per microliter. When the primer concentrations are much greater than the concentration of target, second-order dynamics reduce to pseudo first order. The percentage of annealed template at any time only depends on the primer concentration. So if you double the primer concentration, the required annealing time is halved.” As an added benefit, shorter annealing times increase specificity (4 ). Similar logic goes for extension. You increase the concentration of polymerase, which otherwise becomes
Technology Corner
Fig. 1. Extreme PCR can increase the PCR cycle speed by 1–2 orders of magnitude by increasing the primer and polymerase concentrations. A 4 –water bath system is shown, including annealing, extension, and denaturation temperatures, in addition to a fourth water bath that could be used for reverse transcription, enabling RNA virus amplification. ©Reproduced with permission from C.T. Wittwer.
rate limiting. And with the cost of polymerase falling like crude oil in early 2015, adding a lot of the enzyme becomes very feasible. In a recent report, Wittwer shows efficient and specific DNA amplification of a 45-bp sample with a 26-fold reduction in time when the primer concentration is increased 20-fold and the polymerase concentration is increased 16-fold (5 ). His most aggressive result is a 60-bp amplification with 35 cycles in 14.7 seconds. What about nonspecific binding? The prevailing idea is that high concentrations of primers and polymerase befuddle the reaction because the excess reactants produce nonspecific products. Not true when the reaction runs at extreme speeds, says Wittwer. “Just hit the temperatures precisely.” When the reaction is really fast, there is no time for nonspecific binding or extra polymerase extension. He accomplishes the required speeds by using 2 large water baths to rapidly change the temperature. As noted in this journal, this design is “harking back to the early days of PCR” (6 ). Finally, denaturation “is so fast that it is hard to measure and does not require any chemistry modification.” Wittwer estimates that denaturation occurs in less than 100 milliseconds and is using stopped-flow instrumentation to further pin down denaturation rates. Where Can This Technology Fit In the Field? Wittwer’s vision is a less than 5-minute, end-to-end test that includes sample preparation and detection. “That is
Fig. 2. Wittwer’s 4 –water bath system.
what interests me and excites me. You have to wait a bit, but certainly not the hour. If we can push it down to this time range, more people will use it. I have 100% conviction that the PCR part can be done in under a minute for targets of less than 100 base pairs.” Also, some analysis such as gel electrophoresis or sequencing is required after the PCR. “What I like the best is simple melting analysis. The advantage is that you do the analysis on the same instrument,” he says, having recently demonstrated serial PCR and high-speed melting analysis (7 ). At a melting rate of 0.5 °C per second, “if we need to cover a 30 °C range, that’d require 60 seconds.” “The harder piece and the reason you don’t see 5-minute sample-to-answer PCR systems out on the market yet is that the sample preparation piece depends on the sample matrix; it is hard to provide a very fast one-stop solution that will process every sample type.” However, Wittwer believes, “you can have different front ends that handle different samples. For Ebola you would run blood; a complex matrix that is nevertheless doable.” He admits, “Integrating very fast sample preparation, amplification and analysis in a convenient and user friendly system is not there yet.” “Microfluidic approaches are promising, and perhaps with augmented chemistry, will succeed,” says WitClinical Chemistry 61:4 (2015) 675
Technology Corner twer. Although he has shown 90%–100% PCR efficiency, concerns about the robustness of extreme PCR and whether it can quantify as well as slower systems remain. “The instruments we have developed are not yet practical—we are using large water baths to change temperature quickly.” Poon shares these reservations. “The main concern for this approach is this is still a PCR test, the prototype in the manuscript has 2 water baths only. So this might not be able to do a 1-step RT-PCR [reversetranscription PCR] for emerging RNA viruses.” But Poon is optimistic about Wittwer’s latest breakthrough. “Clearly, this is going in the right direction,” he says. It turns out that Wittwer has already made a 4 – water bath system (Fig. 2), so at least this concern may already be solved.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisi-
676
Clinical Chemistry 61:4 (2015)
tion of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article. Authors’ Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.
References 1. WHO. Urgently needed: rapid, sensitive, safe and simple Ebola diagnostic tests. http:// www.who.int/mediacentre/news/ebola/18-november-2014-diagnostics/en/ (Accessed January 2015). 2. Mullis KB, Ferre´ F, Gibbs RA, editors. The polymerase chain reaction. Boston: Birkhäuser; 1994. 3. Wittwer CT, Fillmore GC, Garling DJ. Minimizing the time required for DNA amplification by efficient heat transfer to small samples. Anal Biochem 1990;186:328 –31. 4. Wittwer CT, Garling DJ. Rapid cycle DNA amplification: time and temperature optimization. Biotechniques 1991;10:76 – 83. 5. Farrar JS, Wittwer CT. Extreme PCR: efficient and specific DNA amplification in 15– 60 seconds. Clin Chem 2015;61:145–53. 6. JF Mackay. Taking it to the extreme: PCR at wittwerspeed. Clin Chem 2015;61:4 –5. 7. Sundberg SO, Wittwer CT, Howell RM, Huuskonen J, Pryor RJ, Farrar JS, et al. Microfluidic genotyping by rapid serial PCR and high-speed melting analysis. Clin Chem 2014; 60:1306 –13.
Extreme PCR: A Breakthrough Innovation for Outbreaks? Vikram Sheel Kumar1* and Molly Webster2
The latest Ebola outbreak is the diagnostics grand challenge of the moment. Groups across the world are racing to build the “ideal test” that according to the WHO should work without laboratory infrastructure, take fewer than 3 steps, report results in under 30 min, and have minimum biosafety requirements (1 ). Leo Poon, head of Public Health Laboratory Sciences at the University of Hong Kong School of Public Health, who was one of the first to decode the SARS (severe acute respiratory syndrome) virus and develop a test for H1N1 influenza, would add “affordable” to that list. “What we need in the field is something simple; and it must be cheap,” he says. What is the most radical innovation with the potential to deal with viral outbreaks? “The portable PCR machine,” Poon shares. Seven thousand miles away a pathologist in Utah may have hit on an innovation that will change PCR forever. Dr. Carl Wittwer, professor of pathology at the University of Utah Medical School, has been recognized by the inventor of PCR, Kary Mullis, as “someone who has thought about PCR in a way that very few others have” (2 ). Wittwer invented the LightCycler system, which was licensed to Roche with over 10 000 units placed across the world. He spoke with this journal about his latest innovation, “extreme PCR.” Why Is This Innovation Important?
Carl Wittwer
“Once a technology becomes established, further innovation is still possible.” says Wittwer, referring to PCR. The dependence on central laboratory testing constrains the ability for a community to deal with an outbreak such as Ebola. The motivation for decentralization is to obtain faster results to provide medical action
1 Boston Children’s Hospital, Boston, MA; 2 Science writer and producer, Brooklyn, NY. * Address correspondence to this author at: Boston Children’s Hospital, 390 Commonwealth Ave., Apt. 605, Boston, MA 02215. Fax 617-899-8944; e-mail [email protected]. Received January 14, 2015; accepted January 16, 2015. DOI: 10.1373/clinchem.2014.236950 © 2015 American Association for Clinical Chemistry
674
Clinical Chemistry 61:4 (2015)
in the field. “That is a place where historically PCR has not been very competitive because of the time required just to amplify—an hour or two. When you show you can amplify in less than a minute, now you can do nucleic acid testing in a very short time,” explains Wittwer, whose rapid-cycle PCR technology that brought PCR time down to 10 –30 min dates back to the 1990s (3 ). “A number of advances including microfluidics showed that PCR in less than 10 minutes was feasible, but the quality of amplification became poor when limited with time,” says Wittwer. “We changed the chemistry of the reaction.” How Does It Work? “This is pretty obvious, and I kick myself for not doing it 20 years ago. The information was all there,” he shares. PCR has 3 steps: annealing, extension, and denaturation. The “critical thing” in PCR is getting the solutions to the required temperatures. Conventional instruments do this poorly, and have to Leo Poon add additional holding times in order for the sample to reach temperature targets. But according to Wittwer, a trick was needed in chemistry, not instrumentation (Fig. 1). Annealing occurs between primers and singlestranded DNA targets. Because there are 2 oligonucleotides binding, it is a “second-order issue.” However, “for most of PCR you have millions of copies of primers per microliter. When the primer concentrations are much greater than the concentration of target, second-order dynamics reduce to pseudo first order. The percentage of annealed template at any time only depends on the primer concentration. So if you double the primer concentration, the required annealing time is halved.” As an added benefit, shorter annealing times increase specificity (4 ). Similar logic goes for extension. You increase the concentration of polymerase, which otherwise becomes
Technology Corner
Fig. 1. Extreme PCR can increase the PCR cycle speed by 1–2 orders of magnitude by increasing the primer and polymerase concentrations. A 4 –water bath system is shown, including annealing, extension, and denaturation temperatures, in addition to a fourth water bath that could be used for reverse transcription, enabling RNA virus amplification. ©Reproduced with permission from C.T. Wittwer.
rate limiting. And with the cost of polymerase falling like crude oil in early 2015, adding a lot of the enzyme becomes very feasible. In a recent report, Wittwer shows efficient and specific DNA amplification of a 45-bp sample with a 26-fold reduction in time when the primer concentration is increased 20-fold and the polymerase concentration is increased 16-fold (5 ). His most aggressive result is a 60-bp amplification with 35 cycles in 14.7 seconds. What about nonspecific binding? The prevailing idea is that high concentrations of primers and polymerase befuddle the reaction because the excess reactants produce nonspecific products. Not true when the reaction runs at extreme speeds, says Wittwer. “Just hit the temperatures precisely.” When the reaction is really fast, there is no time for nonspecific binding or extra polymerase extension. He accomplishes the required speeds by using 2 large water baths to rapidly change the temperature. As noted in this journal, this design is “harking back to the early days of PCR” (6 ). Finally, denaturation “is so fast that it is hard to measure and does not require any chemistry modification.” Wittwer estimates that denaturation occurs in less than 100 milliseconds and is using stopped-flow instrumentation to further pin down denaturation rates. Where Can This Technology Fit In the Field? Wittwer’s vision is a less than 5-minute, end-to-end test that includes sample preparation and detection. “That is
Fig. 2. Wittwer’s 4 –water bath system.
what interests me and excites me. You have to wait a bit, but certainly not the hour. If we can push it down to this time range, more people will use it. I have 100% conviction that the PCR part can be done in under a minute for targets of less than 100 base pairs.” Also, some analysis such as gel electrophoresis or sequencing is required after the PCR. “What I like the best is simple melting analysis. The advantage is that you do the analysis on the same instrument,” he says, having recently demonstrated serial PCR and high-speed melting analysis (7 ). At a melting rate of 0.5 °C per second, “if we need to cover a 30 °C range, that’d require 60 seconds.” “The harder piece and the reason you don’t see 5-minute sample-to-answer PCR systems out on the market yet is that the sample preparation piece depends on the sample matrix; it is hard to provide a very fast one-stop solution that will process every sample type.” However, Wittwer believes, “you can have different front ends that handle different samples. For Ebola you would run blood; a complex matrix that is nevertheless doable.” He admits, “Integrating very fast sample preparation, amplification and analysis in a convenient and user friendly system is not there yet.” “Microfluidic approaches are promising, and perhaps with augmented chemistry, will succeed,” says WitClinical Chemistry 61:4 (2015) 675
Technology Corner twer. Although he has shown 90%–100% PCR efficiency, concerns about the robustness of extreme PCR and whether it can quantify as well as slower systems remain. “The instruments we have developed are not yet practical—we are using large water baths to change temperature quickly.” Poon shares these reservations. “The main concern for this approach is this is still a PCR test, the prototype in the manuscript has 2 water baths only. So this might not be able to do a 1-step RT-PCR [reversetranscription PCR] for emerging RNA viruses.” But Poon is optimistic about Wittwer’s latest breakthrough. “Clearly, this is going in the right direction,” he says. It turns out that Wittwer has already made a 4 – water bath system (Fig. 2), so at least this concern may already be solved.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisi-
676
Clinical Chemistry 61:4 (2015)
tion of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article. Authors’ Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.
References 1. WHO. Urgently needed: rapid, sensitive, safe and simple Ebola diagnostic tests. http:// www.who.int/mediacentre/news/ebola/18-november-2014-diagnostics/en/ (Accessed January 2015). 2. Mullis KB, Ferre´ F, Gibbs RA, editors. The polymerase chain reaction. Boston: Birkhäuser; 1994. 3. Wittwer CT, Fillmore GC, Garling DJ. Minimizing the time required for DNA amplification by efficient heat transfer to small samples. Anal Biochem 1990;186:328 –31. 4. Wittwer CT, Garling DJ. Rapid cycle DNA amplification: time and temperature optimization. Biotechniques 1991;10:76 – 83. 5. Farrar JS, Wittwer CT. Extreme PCR: efficient and specific DNA amplification in 15– 60 seconds. Clin Chem 2015;61:145–53. 6. JF Mackay. Taking it to the extreme: PCR at wittwerspeed. Clin Chem 2015;61:4 –5. 7. Sundberg SO, Wittwer CT, Howell RM, Huuskonen J, Pryor RJ, Farrar JS, et al. Microfluidic genotyping by rapid serial PCR and high-speed melting analysis. Clin Chem 2014; 60:1306 –13.
