As the biotech industry races ever onward with advances in cellomics, proteomics, and genomics, Polymerase Chain Reaction (PCR) remains the gold standard assay and a backbone of the life sciences. PCR can be used as an endpoint assay (qPCR, dPCR, etc.) for study and it can also be used to amplify product for subsequent studies.

Regardless of what a laboratory is using PCR for, the techniques behind PCR are constantly evolving. In this blog, we’ll examine where PCR technology is heading and the research driving it.

Where We’ve Been

Classic PCR applications have typically relied on using microliters (mL) of a given master mixture to produce enough usable material. The mixture and original DNA were combined by hand using micropipettes or simple multipipetters.

However, as demands for genetic testing and research have increased, manual processes have not been able to keep up. Automation and miniaturization are necessary to scale PCR assays to meet demand.

What is currently achievable?

Non-contact, low volume dispensing systems can dispense volumes from microliters (µL) down to picoliters (pL). Primers, samples, and mastermix can be printed into either existing or novel devices and BioDot’s experts are commonly seeing anywhere from 100 to 1000-fold increases to the 96-well format.  High density well plates, microfluidics, and novel slide or chip-based formats are producing 10’s to hundreds of thousands of PCR reactions on a single device.

Miniaturization

When it comes to PCR, processing the assay and data collection are often the bottlenecks. Labs are always looking to assemble more assays in a single run to save time.

Miniaturization is the answer to this problem. In the life sciences, miniaturization of routine assays is shaping every discipline for a few key reasons: Smaller assay volumes decrease costs and increase throughput.

The early days of COVID-19 showed that the standard 96-well PCR was untenable for a problem that required true scale. The first 96-well plate COVID assays were limited to 24 samples per run; With 1-2 hour thermocycle runs, a lab was limited to 100-200 samples per thermocycler per 8 hour shift. It was obvious in the first few months of the pandemic that scale could only be achieved through miniaturization.

Throughput versus Flexibility

Closed systems using emulsion PCR (emPCR) have had an undeniable and profound impact on throughput for PCR. They can produce reactions in nanoliter (nL) volumes at thousands of reactions per second and avoid chimeric molecules. As a result, an impressive library of assays and protocols have been built to take advantage of the emPCR format. But there’s a catch to this – emPCR comes with many constraints on what you can manipulate or adjust.  If a researcher is looking to tweak or adjust the assay in ways that exceed the capabilities of the pre-defined assays (adding new and novel labeling strategies, adding processing steps to expand data collection, changing measurement/reading technologies, etc.), they will likely need to move to an open platform to achieve the customization they need. In other words, jailbreaking a closed system is much easier said than done.

Assays enjoy greater flexibility when conducted in an open format. With total PCR reaction volumes below 1µL (10-500nL individual dispenses of mastermix, primers, and sample), dispensing into small/open wells with a liquid handler allows for unique combinations of reagents. Additionally, the sequence and timing of any additive can be fine-tuned to optimize protocols. Lastly, open wells provide the opportunity to alter parameters for specific wells if you would like to extract the PCR products for another assay or protocol.  

While there are other considerations, closed systems offer the highest throughput, while open systems provide the greatest flexibility.  Both have delivered incredible savings with respect to per assay cost.

Other Considerations

As reaction volumes for PCR decrease below 10µL, precision in dispense volume becomes increasingly critical. Quantitative techniques and statistical analyses require precision while still maintaining throughput.

No Touching!

Non-contact methods of deposition are more precise than contact or semi-contact methods. The properties of surface materials that interact with target mixture will influence dispense volumes. Even small differences in charge can draw varying volumes from a dispense tip. Thus non-contact dispensing systems and techniques will produce more reliable results, provided that CV’s below 1µL are below 5%.

Another benefit of non-contact deposition is that chemistries can be delivered faster than contact/near-contact methods. For contact deposition methods, it takes time to pause, move to the surface for each dispense, then move away from the surface. This can be a cumbersome and slow process and it compounds with each dispense cycle. Long dispense cycles may artificially create variation when reagents are deposited due time lags between the first and subsequent dispenses.

How Low (or High) Can You Go?  

The dynamic range of the dispensing system is also important to consider in PCR. In some instances, it may be desirable to dispense PCR constituents at ratios greater or smaller than 1:1. If that is the case, you need a system that has the flexibility to dispense microliters AND picoliters.

Additionally, not all dynamic ranges are created equal. Any dispenser that can dispense picoliters can also dispense microliters or even milliliters if it repeats the process a few hundred times. While this can be sufficient for some applications, it is often slow. Additionally, the CV of each individual drop can compound as more droplets are added, leading to a greater overall CV.

Dude, Where’s My Drop?

When working with miniaturized assays, a key consideration is evaporation. Can your reagents maintain performance when dried? If not, reducing the time to dispense is critical. Alternatively, the rate of evaporation can be manipulated through other means. Chilling reagents, increasing humidity, or using a sealing system (e.g., foils, plastics, oils) are all possible options to slow or prevent evaporation.

Final Thoughts

Whether you are looking at closed systems or open platforms, the future of PCR (and genomics exploration in general) is bright. Thousand-fold increases in throughput are real and the tools to build sub-microliter and sub-nanoliter assays are here and ready to be deployed.

As with most things, miniaturized technics are not a ‘one size fits all’. Consider the pros and cons of any platform carefully and reach out to partners early to avoid missteps and setbacks.

About the Author

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Brian Kirk is the VP of Strategy and Product Development at BioDot™. Brian has been with BioDot since 2001 and has spent that time designing, developing, and marketing high throughput manufacturing systems for many of the world’s leading diagnostic and life science companies. Today, Brian now leads the Business Development team within BioDot where he leverages BioDot’s expertise in high throughput nanoliter and picoliter printing technologies to commercialize important tools for emerging life science markets.