Extreme PCR

PCR is a vital technology used in molecular biology and diagnostics. Temperature cycling creates a series of events : DNA denaturation, primer annealing, and polymerase extension. These stages of PCR result in exponential DNA amplification.

When PCR was discovered in 1985, each cycle took several minutes to perform, requiring hours for completion. Since then, many groups have developed unique methods to make PCR faster (read more about Rapid-cycle PCR). In the early 90's, Rapid-cycle PCR was developed with 10 - 30 second cycles. Groups have aimed at 10 second cycles (2, 3), 5.25 - 4.6 second cycles (4, 5), 3 second cycles (6, 7) using various technological advances, and 2.7 second cycles (8) using gallium and glass capillaries.


Unfortunately, these attempts to reduce PCR cycle times often used simple targets and compromised efficiency and yield. Practical sub 5 minute PCR amplification of genomic DNA proved difficult.

Our objective of cycling at extreme speeds (0.4 to 2.0 seconds per cycle) required innovative instrumentation and increased concentrations of reagents (primer and polymerase concentration) to ensure not only speed but high efficiency and yield. The resulting technique, Extreme PCR (Published in Clinical Chemistry), demonstrates the feasibility of while-you-wait testing for tests where immediate results are critical.

To perform Extreme PCR:

• Instrumentation for temperature cycling 1- to 5-µL samples in 0.4-2.0 seconds used physical translation of samples in capillary tubes into water baths of different temperatures with a stepper motor.
• Annealing / extension was performed at 62°C - 76°C with denaturation temperatures of 85°C - 92°C.
• Targets (single-copy genes from human genomic DNA) ranged in length from 45- to 102-bp.
• The time required for PCR is inversely related to the concentration of critical reactants. By increasing primer and polymerase concentrations 10- to 20-fold with temperature cycles of 0.4-2.0 s, specific, high-yield PCR with an efficiency of >90% was possible from human DNA in 15-30 seconds.

Compared to rapid cycle PCR with a 20 second cycle (30 cycles in 10 minutes), an extreme PCR cycle can be completed in less than 1 second, over 20 times faster (30 cycles in <30 seconds). The figure below shows the sample temperature trace during extreme and rapid cycle PCR.



Extreme cycling speeds are obtained by movement of samples contained in capillaries into water baths of different temperatures by rotation of a stepper motor. Fluorescence is monitored at the tip of the capillary with a fiber optic.

First Real Time Extreme PCR Prototype

Later designs included more water baths and stepper motors for 2-axes. Instead of flipping between water baths, the samples traced arcs between the different baths.

Below are agarose gel results for extreme and rapid cycle PCR. The only differences between the reactions were cycling speed and the concentrations of polymerase and primers. The extreme reactions have 10-20-fold the amount of polymerase and primers compared to the rapid cycle reactions.

Melting curves of the products and no template controls (NTC) are also shown. Note that the Tm of the product from the extreme reaction is lower because of a higher glycerol concentration from the larger volume of polymerase.




Incorporation of optics allowed monitoring PCR in real time as shown in the following videos:






Instead of using water baths, extreme PCR was combined with melting analysis on a microfluidic instrument in less than 60 seconds (Myrick et al. 2019).



Instead of plotting temperature vs time or fluorescence vs time as in the above traces, plots of fluorescence vs temperature reflect the extent of hybridization when dsDNA dyes are used. The traces below compare fluorescence vs temperature plots of rapid cycle vs extreme PCR.




Speed does not compromise extreme PCR. The sensitivity and efficiency of 2 extreme PCRs are shown below:

Extreme PCR Efficiency and Specificity

To our knowledge, the fastest PCR ever performed was analyzed on a gel. The amplification of a 60 bp genomic fragment was completed in just under 15 seconds, resulting in a specific, strong band after 35 cycles.




Finally, we demonstrated the potential for integrated molecular diagnostics in less than 1 minute, starting with a finger prick and ending with real-time amplification, as shown in the following video:

1. Wittwer et al. (2025) Int J Lab Hematol manuscript Pubmed
2. Wittwer (2023) Methods Mol Biol manuscript Pubmed
3. Myrick et al. (2019) Nucleic Acids Res manuscript Pubmed
4. Millington et al. (2019) Biomol Detect Quantif manuscript Pubmed
5. Jafek et al. (2018) Anal Chem manuscript Analytical Chemistry
6. Trauba & Wittwer. (2017) J. Bio S & E manuscript JBSE
7. Farrar & Wittwer (2015). Extreme PCR Clinical Chemistry
8. J Mackay (2014) Clin Chem manuscript Pubmed