Raising the Bar on PCR

For more than 30 years, PCR has been a true cornerstone of molecular biology. The simplicity with which it can amplify DNA makes it an essential step in many cutting-edge genomics applications. Find out what today’s scientists expect when performing PCR in terms of accuracy, consistency, and speed.

Polymerase chain reaction (PCR) is the most commonly used technique for creating copies of a DNA fragment in the laboratory. Soon after its development, its speed, combined with the fact that it can be easily automated, provided the impetus for large-scale genomics studies, such as the human genome project, which spanned most of the 1990s. As DNA sequencing techniques evolved and next-generation sequencing became the norm in the 21st century, PCR remained an integral part of the workflow.

Of course, sequencing is only one application where PCR plays this key role. Researchers also use the technique extensively in cloning, DNA quantification (through quantitative PCR, qPCR), and gene expression analysis (e.g. through reverse transcriptase PCR, RT-PCR). In clinical applications, PCR forms the basis of many molecular diagnostic tests.

Ready, steady, amplify

Although the process of DNA amplification by thermal cycling is straightforward and easy to automate, setting up a PCR run often remains a time-consuming affair. PCR setup involves a high number of pipetting steps, which not only increases the time of the experiment, but also the risk of errors.

Setting up a PCR run involves mixing all the different samples with the ingredients needed for DNA amplification, including nucleotides, DNA polymerase, primers, and so on. Many types of kits are commercially available to simplify this process, but often it still requires manual pipetting. Due to the nature of PCR work, even small experiments can take up most of a 96-well plate, requiring hundreds of pipetting steps.

Successful PCRs today

Because of the central role that PCR plays in today’s research environment, demands on the PCR setup are tight. First and foremost, of course, there’s accuracy; pipetting in PCR setup routinely involves transferring microliter, or even sub-microliter, volumes, which is associated with a high error rate and therefore the risk of inaccurate results. Speed is also of the essence. Regardless of the scale of an experiment or test, anything that can speed up the PCR workflow is highly beneficial in a busy laboratory environment.

Finally, there is the reliability of the PCR run. PCR is notoriously sensitive to contamination from external factors and sample degradation. With this in mind, scientists have to ensure that equipment and consumables are not just clean, but that they are ‘PCR clean’ (i.e. free of DNA, DNase, RNA, RNase, and PCR inhibitors). Also, to combat sample degradation, it is important in most cases to keep samples as cool as possible while preparing a PCR plate. With manual preparation, scientists often achieve this by placing the plates and reagents on ice.

Automation all the way

One way of tackling the challenges of PCR is to extend automation to the setup phase of the experiment. The use of an automated liquid handling system provides a range of benefits to lab users and is an attractive solution – not just for high-throughput use, but also for smaller-scale PCR work.

Automated liquid handling greatly reduces time spent setting up PCR plates. These time savings not only relate to total setup time, but also hands-on time. For example, automated pipetting makes it possible set up a 96-well plate with 32 different samples in three replicates in 10 minutes.

When it comes to accuracy, a fully automated approach helps to create a higher level of certainty about pipetting volume. Modern liquid handling systems are calibrated for consistent dispensing and independent testing can be carried out to determine the coefficient of variation of the equipment at different volumes.

The use of an automated liquid handling approach also helps in preventing contamination and degradation of sample DNA. With automated handling, all pipetting takes place in a fully enclosed space, reducing the risk of contamination from external sources. The use of cooling elements also makes it easier to keep all samples and reagents at a constant, low temperature so plates do not have to be put on ice during the setup.

Eppendorf offers a range of products that can support and automate PCR workflow.  These include the epMotion® 5070 automatic liquid handling system with a special, newly launched PCR adaptor, the Mastercycler® X50, and Eppendorf twin.tec® LoBind PCR plates. These advanced solutions help to ensure accurate PCR results with low risk of contamination in a fast, efficient workflow.

To find out more about how we make sure automated pipetting is guaranteed within specifications, read our application note on evaluating the epMotion pipetting tool using the Artel® MVS® multichannel verification system.

Artel® and MVS® are registered trademarks of Artel Inc., USA. epMotion®, Mastercycler® and Eppendorf twin.tec® are registered trademarks of Eppendorf AG, Germany.