Taking it to the extreme: PCR at wittwerspeed.

If the start times of PCR thermal cycling were plotted relative to frequency, we could expect to easily see sharp peaks of start times immediately before morning/afternoon coffee breaks, lunch times, and end of day, by operators safe in the knowledge that the amplification would take 1–2 hours to complete. Perhaps it is a little faster in many, but not most, diagnostic laboratories. Indeed, careful experimental planning when I was a student allowed leisure activities at opportune times “while the PCR was running.” At the time, these PCRs were typically 50–100 L in volume and used relatively thick 0.5-mL tubes. In the late 1990s, the Guinness Book of World Records held an entry for “fastest DNA fingerprint” of approximately 11 min (essentially rapid real-time PCR detection). Further demonstrating that faster amplification concepts are not new, the term “rapid-cycle PCR” was coined in the early 1990s to define a thermal cycling program performed in 10–30 min (1 ). The Guinness entry and rapid-cycle PCR have a common denominator: the world record was held by the original capillary-based LightCycler, and similar capillaries were used for the rapid-cycle PCR—both in the hands of Carl Wittwer from the University of Utah. The work of Professor Wittwer and his team is no stranger to these pages, as they previously published realtime PCR applications on the LightCycler (2) as well as early papers on the now well-adopted genotyping and variant scanning methodology of high-resolution melting analysis (HRMA) developed in his laboratory (3, 4)—work that more than justified Wittwer as the subject of an “Inquiring Minds” article, also in Clinical Chemistry (5). Since these developments, he has returned to his first molecular interest: increasing the speed of PCR. Total PCR analysis time has decreased largely because of the advent of real-time PCR and now digital droplet PCR, in which gel electrophoresis bands have been replaced with amplification curves and scatter plots. Yet the major time component in the PCR itself is the thermal cycling time. Recent efforts to decrease these times have used chipbased reactions using microvolume reactions in 6 min (6) or a rapid-cycling instrument with high convective heat transfer in 3 min (7). New companies are heating the reactions on particles (rather than heating reaction volumes) as a means to increasing ramp rates (the speed at which reactions heat and cool among the various temperature cycles) (8). Despite these newer methods, most have yet to reach the commercial market, and none have come close to penetrating the dominance of the slower Peltier-based technology. Recent work in Wittwer’s laboratory has characterized the effect of PCR buffer and additive components on DNA polymerase activity using a novel nonradioactive assay (9 ), thus paving the way for further improvements in PCR. In this issue of Clinical Chemistry, Farrar and Wittwer return to these original reaction capillaries (thinner than the current LightCycler capillaries and thus allowing faster heat transfer to the reaction volumes) and describe their concept of Extreme PCR, pushing down the time of amplification to 15 s—as little as 14.7 s for 35 cycles, in fact (10 ). Such short times demand better accuracy. Having frequently challenged the dogmas that existed in PCR and DNA melting kinetics, Wittwer’s laboratory has overturned the belief that higher primer and enzyme concentrations always lead to nonspecific amplifications. That belief is true if typical thermal cycling rates are used; but under extreme PCR conditions, the amplifications result in sharp specific bands (and melting peaks), presumably because of a lack of time for off-target products to be generated. This increased specificity was also observed for certain targets in the original rapid-cycle PCR of 10–30 min compared with thermal cycling of 2–3 h (1 ). Extreme PCR has also used existing enzymes successfully, as well as newer-generation highly processive systems. These current enzyme systems have been successfully used previously in reactions of 3 min (7 ). Thus the extreme PCR technique requires 3 main parameters to ensure the robust, efficient, and sensitive amplifications we typically enjoy: rapid temperature cycling (very rapid!), higher primer concentrations (typically 20-fold higher than what would be considered usual), and higher enzyme concentrations (approximately 15-fold higher). Standard enzymes are less expensive now due to expiration of the Taq patents, and thus the chemistry modifications of extreme PCR look to have little dnature diagnostics & research, Gisborne, New Zealand. * Address correspondence to the author at: dnature, 24 Island Rd., Gisborne 4010, New Zealand. E-mail [email protected]. Received October 27, 2014; accepted October 30, 2014. Previously published online at DOI: 10.1373/clinchem.2014.233338 © 2014 American Association for Clinical Chemistry Clinical Chemistry 61:1 4–5 (2015) Editorials