Under-three minute PCR: Probing the limits of fast amplification

Abstract

Nucleic acid amplification is enormously useful to the biotechnology and clinical diagnostic communities; however, to date point-of-use PCR has been hindered by thermal cycling architectures and protocols that do not allow for near-instantaneous results. In this work we demonstrate PCR amplification of synthetic SARS respiratory pathogenic targets and bacterial genomic DNA in less than three minutes in a hardware configuration utilizing convenient sample loading and disposal. Instead of sample miniaturization techniques, near-instantaneous heating and cooling of 5 μL reaction volumes is enabled by convective heat transfer of a thermal fluid through porous media combined with an integrated electrical heater. This method of rapid heat transfer has enabled 30 cycles of PCR amplification to be completed in as little as two minutes and eighteen seconds. Surprisingly, multiple enzymes have been shown to work at these breakthrough speeds on our system. A tool for measuring enzyme kinetics now exists and can allow polymerase optimization through directed evolution studies. Pairing this instrument technology with modified polymerases should result in a new paradigm for high-throughput, ultra-fast PCR and will hopefully improve our ability to quickly respond to the next viral pandemic.

Extreme PCR: Efficient and Specific DNA Amplification in 15–60 Seconds

“…Recently, systems using hot and cool water for heat transfer through metal blocks have achieved 5.25-and 4.6-s cycles (8,9 ). Infrared-mediated temperature cycling demonstrated 3-s cycles (10 ); vapor pressure-augmented, continuous-flow PCR, 3-s cycles (11 ); and PCR within glass capillaries submerged in heated and cooled liquid gallium, 2.7-s cycles (12 ).…”

Taking It to the Extreme: PCR at Wittwerspeed

“…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 Ͻ3 min (7 ).…”

“…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 Ͻ6 min (6 ) or a rapid-cycling instrument with high convective heat transfer in Ͻ3 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 ).…”

The Development of Direct Ultra-Fast PCR for Forensic Genotyping Using Short Channel Microfluidic Systems With Enhanced Sieving Matrices

“…120,169,170 Results can be further speeded up by upgrading the chemistry of the Taq enzyme to permit high-speed amplification of DNA, reducing the overall reaction time from 2 ½ hours to under 15 minutes. 123,171 Lastly, STR analysis using short channel microfluidic systems such as the system developed in Chapter 9 can be achieved in less than 2 minutes. Combining these three processes would result in an ultra-fast, sample to genotype result in about 20…”

“…Infra-red (IR) radiation or lasers pulses have also been used to heat samples. 171 Generally, the use of cycling blocks made from highly conductive alloys permits a more rapid transfer of heat. Most high-speed thermal cyclers use this technique, as it is particularly effective for high-speed cooling.…”