Principles of 'Fast' PCR

Polymerase chain reaction (PCR) is a powerful technique used to amplify specific fragments of DNA sequences of interest through repeated thermal cycling. For amplification to be effective, the PCR machine must be able to achieve and maintain certain temperatures for specified durations. These temperature dependent steps include, typically, between 90-94°C for the denaturation of the double-stranded (ds) DNA, 50-70°C for annealing of the primers to the single stranded (ss) DNA, and 70-75°C for enzymatic extension of the primers.

In general, a traditional PCR takes around 2 hours, though this does not include sample preparation, subsequent analysis, and or the desired number of amplification cycles. Though the denaturation and renaturation steps can be performed in just a few minutes, PCR cycle times are ultimately limited by the elongation step which is ultimately dependent on the length of the amplified product.

While conventional PCR can provide sufficient quantities of material for detection even at the single copy level, fast reporting of the results can be severely hindered. Rapid reporting of PCR results is particularly important in applications such as the detection of pathogens, for example the SARS-CoV-2 virus, or for real-time molecular forecasting before a sensitive surgical procedure.

A variation of the technique, called rapid or fast PCR, uses specialized enzymes and reaction conditions to amplify DNA in a quicker, timely manner, when compared to traditional PCR. In general, rapid PCR techniques overcome time limitations by removing the need for excessive thermal cycling. Instead, rapid PCR utilized systems that contain small capillary-channeled vessels with a lower thermal capacitance which allows for faster heat transfer.

A rapid PCR test is so quick and sensitive that it has been used for point of care testing, and can be performed in a medical office or pharmacy using desktop machines without the need to send the sample to a highly complex laboratory. These tests are used, more simply, to confirm the presence or absence of viral RNA in a sample where extensive DNA amplification is not necessary. Rapid PCR tests are often compared to rapid antigen tests due to their quick testing times. Importantly though, rapid PCR tests are more accurate than antigen tests as they identify specific genetic material from a pathogen, and not just specific sugars or proteins on an antigens surface.

Outside of point of care diagnostic testing, rapid PCR has various applications and has been used in forensics, paternity testing, to diagnose genetic disorders as well as cancer. Additionally, it can be used to study gene expression, regulation, and function, or as a quality control step for pharmaceuticals and biologics. Though rapid PCR tests offer fast results and reduce the time required for amplification, PCR product yields will be decreased, and results may have lower sensitivity or accuracy compared to standard PCR. Rapid PCR tests, similar to traditional PCR, may suffer from false-positive and false-negative results, so a secondary test is often needed to confirm results.

 

 

 

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Original created on January 24, 2024, last updated on January 24, 2024
Tagged under: pcr, DNA, amplification