PCR is a nearly ubiquitous, reliable, and powerful technology found in most molecular biology labs. It is used to amplify specific stretches of DNA for genotyping, cloning, analysis of single nucleotide variations, for example, and even serves as the basis for most next-generation sequencing (NGS) preparation. While it is now, for the most part, a mature technology, improvements and innovations—some disruptive, some niche, and some incremental—have certainly been seen in the three-plus decades since the polymerase chain reaction was first introduced. Here we look at some of its more recent iterations you may not have encountered in graduate school.
qPCR
Quantitative PCR (qPCR) is PCR that incorporates fluorescence—either as intercalating dyes or sequence-specific probes—into nascent DNA as it is amplified. The accumulation of fluorescent signal is generally tracked as the reaction proceeds, allowing for results to be obtained digitally in real time rather than needing to visualize results by gel electrophoresis. The point at which signal can be distinguished from noise is termed cycle threshold (Ct): comparing Ct values against a standard allows for quantitation of the original target sequence. Samples can be multiplexed by using different fluors for different targets, with some instruments capable of querying at least six colors. qPCR can also be used as an endpoint technology, with the presence or absence of fluorescence at the end of the run as a yes/no answer.
“The qPCR technology has demanded that manufacturers get the chemistry right. So now you have incredibly robust polymerases, priming strategies, and buffers that allow you to do that,” notes Jim Huggett, principle scientist at LGC and senior lecturer at the University of Surrey. That has enabled other PCR formats, such as low volume high-throughput and digital PCR (dPCR), to piggyback on “these reagents that give you good dynamic ranges and high precision measurements.”
High throughput
The term “high-throughput” is generally associated with processing multiple samples efficiently. This can mean many samples simultaneously, or multiple answers from a single sample, or a seamless continuous operation; it can mean fast time-to-result or an end-to-end automated process with little hands-on requirement. Regardless of the precise definition, throughput has trended upwards since PCR meant manually moving a single tube between water baths.
Thermocyclers able to process 96, or even 384 samples are not new. Some can even handle 1536 samples at a time (or in the case of Wafergen’s SmartChip Real-Time PCR System, 5,184). These may be found robotically integrated with components for a streamlined pipeline. Analytik Jena introduced a 384-well plate instrument last year, for example, and “this year we are getting a new one for automated qPCR, which means you can add PCR machines to a liquid-handling system, a pipetting robot, and you can load the plates automatically into this device,” says Melanie Jahn, the company’s real-time product manager.
Many instruments use “pre-made chips or arrays that will have assays pre-dispensed in a certain format or configuration and have to be designed and ordered in advance,” explains Luke Linz, laboratory operations manager at LGC Douglas Scientific. Others, like those built around Douglas Scientific’s continuous polymer Array Tape consumable, “offer more flexibility in terms of ability to change assays on a day-to-day basis if that’s required.”
An added benefit of large arrays is that reaction volume tends to be small, with corresponding savings in reagents and sample. Array Tape’s standard volume is 1.6 microliters, for example, while some formats require even less.
Ahram Biosystems’ Palm PCR S1 real-time qPCR instrument, introduced this past March, is high-throughput in a different sense. By using three distinct heating blocks, it takes a mere 18 seconds per cycle—about 12 minutes total—for amplification and detection of up to 24 samples across six color channels, points out Hyun Jin Hwang, Ahram’s president and CEO.
Array Tape reduces microtitre plates needed, one roll of 768-well Array Tape equals 100 microtitre plates, according to LGC Douglas Scientific.
Portability and point of care
The Palm PCR S1 and its (non-fluorescent traditional endpoint PCR) sibling Palm PCR are stand-alone portable instruments that can run on battery power. Hwang says that there is a lot of interest from countries without reliable electricity, as well as from medical diagnostics sites where time-to-results may be of the essence.
Another option for the field is the Biomeme platform, the center of which is its water bottle-sized two3 instrument that “turns an iPhone into a RT-qPCR device,” says Biomeme’s co-founder and business lead Max Perelman. Coupled with their optimized sample-preparation kits and pre-mixed shelf-stable lyophilized reagents, the low-throughput platform (only three samples at a time) “brings the lab to the sample.” It’s “an awesome piece of engineering that holds up well under real-world field testing, … it’s hard to beat for point of need detection,” notes Jonathan Jacobs, senior advisor at MRIGlobal. Biomeme expects to release its three9 instrument later this year, which will be able to simultaneously test for up to 27 targets (nine samples with three color channels) using either an Android or an iOS mobile device.
But PCR remains for the most part a research and preclinical tool, with a few notable exceptions…
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But PCR remains for the most part a research and preclinical tool, with a few notable exceptions such as rapid microbial testing, virology, and for some cancers, says Huggett. For the point of care (POC) situation, it’s a fairly complex reaction with sometimes better alternatives (such as serology), and a physician will often want to know more than the presence of a gene. That being said, there are some low-throughput push-button POC solutions based on PCR-based platforms, such Cepheid and BioFire, that are gaining traction in clinics and hospital labs.
dPCR
Any discussion about advances in PCR would be inadequate with a mere mention of dPCR. Its division of a PCR reaction into hundreds or thousands of individual reactions—typically in droplets or wells—yields a collection of yes/no answers, and allows for dPCR’s well-known abilities to measure rare variants, perform haplotype analysis, and offer more precision quantification.
Yet for Huggett, although “it’s the least of the sexy things,” dPCR’s reproducibility is the advantage he most likes to tout. Referring to a study he published last year, he concluded that “dPCR could be a major solution to the noise, nonspecificity, and relativity of qPCR. It doesn’t have this issue of an amplification curve and the ambiguities of setting a threshold. It appears to work incredibly reproducibly.”
New entrants such as Stilla join more established vendors such as Bio-Rad, Thermo Fisher Scientific, and Raindance to offer a variety of options in the dPCR marketplace.
Huggett predicts that eventually next-generation sequencing (NGS) will “take over everything.” But when that eventuality will occur, and how much it will still rely on PCR, is still an open question.
Image courtesy of Dreamstime.