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.

Thirty years have passed since PCR was discovered and many researchers have investigated techniques 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 cycles times compromised efficiency and yield as well as used simpler targets. 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 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 was developed to temperature cycle 1- to 5-µL samples in 0.4-2.0 seconds.
• Annealing / extension performed at 62°C - 76°C and denaturation temperatures of 85°C - 92°C.
• Targets (single-copy genes from human genomic DNA) ranged in length from 45- to 102-bp.
• Amplification, as shown in the video below, is captured via real-time optical monitoring.

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, efficient (>90%), specific, high-yield PCR from human DNA is possible in <15 seconds.

• Using single-copy genes from human genomic DNA, we amplified 45- to 102-bp targets in 15-60 seconds.
• Amplification efficiencies were 91.7%-95.8% by use of 0.8- to 1.9-second cycles with single-molecule sensitivity

1. Farrar & Wittwer. Extreme PCR Clinical Chemistry
2. Zhang & Xing (2007) Nucleic Acids Res. manuscript Pubmed
3. Roper et al. (2005) Anal Chem manuscript Pubmed
4. Wheeler et al. (2011) Analyst manuscript Pubmed
5. Terazono et al. (2010) Jpn J Appl Phys manuscript PDF
6. Huhmer & Landers. (2000) Anal Chem manuscript abstract
7. Fuchiwaki et al. (2011) Biosens Bioelectron manuscript Pubmed
8. Maltezos et al. (2010) Appl Phys Lett manuscript CalTech