PCR methodologies have clearly evolved over the years. Although the fundamental procedure – denature, anneal, extend – has not changed much, countless PCR variations have been created and applied to answer unique biological questions. From the engineering of thermostable polymerases and construction of automated thermocyclers, to newer digital PCR (dPCR) methodologies and associated microfluidic devices for handling high-throughput PCR applications, no one can say that PCR is a static method.
When the editors of BioTechniques met to discuss the future of PCR, several exciting new developments from 2012 were noted that should lead to significant advances in many of these fields.
As biologists look to exploring molecular events in single cells, it should come as no surprise that PCR methods have moved toward the goal of examining single molecules. To accomplish this, reaction volumes need to be decreased significantly. Microfluidic PCR systems, with its carefully designed channels and intricate piping and plumbing of solutions, can reduce volumes of reagents, speed the PCR process, and work with very small sample amounts. But small volumes and faster cycling has come at a cost; progress in microfluidic PCR has been slow as researchers dealt with poor PCR efficiency, contamination during the repeated use of the devices, and costly fabrication. Still, recent successes suggest that some of the early bugs have been worked out and this field could be primed to take off in 2013.
In April, Pak et al. published the design and validation of an infrared radiative thermocycler, along with an associated microfluidic chip that allows users to simply load the chip into the instrument and remove it after amplification. Previous systems required repeated calibration steps and thermocouple insertion and alignment, but eliminating these requirements makes infrared microfluidic PCR accessible to laboratories without engineering training (1). Other notable improvements made this year that could have an impact on the way microfluidic PCR is done in 2013 include the development of a semi-reflective, non-reactive platinum nanoparticle monolayer coating for the channels within a microfluidic device that greatly improves interferometry signals for non-contact temperature sensing (2), a new approach for single cell real-time microfluidic PCR (3), and the development of several new specific microfluidic-based point of care diagnostic tests.
Following on the heels of reduced reaction volumes provided with microfluidic PCR, developers have sought to increase throughput and sensitivity using dPCR. In dPCR, picoliter reactions are created by microfluidic or droplet-generating platforms capable of partitioning anywhere from hundreds of thousands to millions of reactions in a single day. Solutions can be diluted such that each droplet or reaction vessel contains either a single template or no template at all, so screening for positive and negative reactions provides absolute quantification without the need for a standard curve. This approach is ideal when studying copy number variation, rare sequence and mutation detection, or gene expression.
While dPCR is still in its early days, studies published in 2012 clearly demonstrate its potential. From comparisons between dPCR and qPCR for template quantification (4) to demonstrating the potential of dPCR to detect specific contaminating organisms and disease biomarkers and the use of a dPCR system for quality control in sequencing library preparation (5), this year we are witnessing the more widespread development and adoption of dPCR. And with the launch in April of a new droplet-based platform by RainDance Technologies that enables multiplexing of dPCR reactions and increases throughput potential up to a billion reactions per day, we can expect even more advances in dPCR in the year to come.
As with PCR methods in the past, incremental steps will be made, with some groups developing new platforms, others optimizing reagents and procedures, and each of these advances eventually coming together to present low input, high-throughput PCR options that are available to researchers in many laboratories as well as those working in field environments.
1. Biomed Microdevices (2012) 14:427–433(Pak et al.)
2. Lab Chip, 2012,12, 127-132
3. Nat Protoc. 2012 Apr 5;7(5):829-38.
4. J Clin Microbiol. 2012 Dec 5.
5. Electrophoresis. 2012 Dec;33(23):3506-13.