From reagents to analytical software, new techniques enhance the amplification of nucleic acids.
Although the polymerase chain reaction (PCR) emerged more than a quarter century ago, it seems like an old friend to molecular biologists. “The technology itself is just elegantly beautiful,” says John Gerace, MBA, vice president and general manager of PCR systems at Life Technologies. It also comes in two main “flavors”: end-point based (PCR) and quantitative real-time PCR (qPCR). Various companies push both versions to new capabilities.
All forms of PCR face some similar obstacles. “The most important challenges for PCR today are reliability of results, as well as reducing the time to finding results and the cost,” says Ulla Deutsch, PhD, senior global product manager amplification at Qiagen. Beyond overcoming those obstacles, this technology is also spreading to a range of new applications.
Prepping the pieces
Like many other assays, the keys to good nucleic-acid amplification begin at the sample-prep stages. “Good lab practices are very important to get reliable PCR results,” says Manju Sethi, product manager for Thermo Fisher Scientific NanoDrop products. In particular, it works best if a researcher knows the starting amounts of nucleic acids. “If you don’t know how much you start with, it could compromise the outcome,” Sethi says.
To get started with a known sample, researchers can use a Thermo Scientific NanoDrop UV-Vis spectrophotometer. As Sethi explains: “It allows you to quantitate and assess the purity of nucleic acids: ‘My DNA is such a concentration and this pure.’"
This micro-volume spectrophotometer also does away with the need for a cuvette. Instead, users put a microliter of sample on a surface. “With NanoDrop, the light only goes through the droplet of sample, so you can directly measure very concentrated samples, eliminating the need for dilutions,” says Sethi. It also uses no consumable materials and delivers a result in less than five seconds.
This instrument is also small, with a footprint less than the size of a sheet of paper. “You could have this on every bench for quality control before PCR,” says Sethi. “Once you have the sample at the right concentration, then bring it to the PCR instrument.”
The selection of probes and quenchers also impacts PCR results. To get a higher signal, researchers can also use the ZEN Double-Quenched Probes from Integrated DNA Technologies (IDT). These provide a quencher near the 5’ fluorophore and another at the 3’ end. “With this approach, the dye is closer to the quenchers, so you get better quenching and less background fluorescence in qPCR,” says Stephen Gunstream, IDT’s vice president of marketing and strategy. “That creates more signal and more sensitivity.”
Size and Time
Researchers want to amplify longer and longer nucleic-acid strings. “Some of the new challenges include finding enzymes that can amplify long sequences with really high accuracy,” says Scott O’Brien, product line manager in the genomics division at Agilent. Moreover, researchers want to run PCR faster. Agilent’s Brilliant III Ultra-Fast QPCR Master Mix meets this need. “Here we used mutagenesis techniques to create a new fast enzyme,” O’Brien explains. The result is a Taq mutant that churns out qPCR results in about half an hour.
The instrumentation behind analysis also makes PCR more efficient. For instance, the LabChip GX from Caliper Life Sciences lets researchers analyze 96- or 384-well plates for end-point PCR. “This system also saves precious samples,” says Isaac Meek, associate director of marketing at Caliper. “It only needs 300 nanoliters per well, because the technology uses microfluidics.” Furthermore, this instrument works fast—analyzing a 96-well plate in under an hour. Meek also points out that simple graphical user interfaces help researchers run assays and even perform some analysis.
Working with smaller sample sizes also drove the development of the GenomePlex Complete WGA and TransPlex Whole Transcriptome Amplification Kits from Sigma-Aldrich. “These kits work with single nanograms of sample and produce products in the microgram region,” says Ernie Mueller, R&D scientist at Sigma-Aldrich.
Beyond working with small samples, these kits work efficiently. “The reagents in these kits activate quickly with antibody-based hot start,” says Christian Reese, product manager for all nucleic acid amplification at Sigma-Aldrich. “The antibody is less damaging, and also more sensitive and accurate.” As Reese points out, this lets these kits be used on samples that have been stored for decades, even if the samples were kept in suboptimal conditions. “So data that have been tracked from a patient by physicians can be tied to data from nucleic-acid analysis on older samples in some cases,” Reese says. “This will help researchers get into the mechanism of diseases and find more molecular markers.”
In today’s labs, workflow also plays a fundamental role in efficiency. “In designing the ViiA 7 Real-Time PCR System, we spent a lot of time looking at what our users do,” says Gerace of Life Technologies. “We looked at the workflow across real-time PCR, from when the instrument shows up on a loading dock to getting it up and running to maintenance.”
This TaqMan-based instrument gives users hardware and software options for efficiency. For example, it can accommodate various multi-well plates, and it includes many software applications, including a drop-down list for ordering supplies. “The whole software architecture has changed,” adds Gerace. “It’s set up for third parties to build applications that interface with their software.”
For example, Life Technologies is promoting the use of this instrument with a robot from Caliper Life Sciences. Life Technologies is also developing applications for data handling. “We’ll set the pace for other companies to develop more applications, such as ones for data analysis,” Gerace says.
SABiosciences, a Qiagen company, also increased the simplicity and speed of PCR with its new RT2 Profiler PCR Arrays. These arrays cover more than 100 pathways, including gene expression related to kidney and liver toxicity.
Solutions for simplification
“With endpoint PCR, lots of people come into labs and start using this technique without much experience,” says Gabriela Saldanha, product manager at Promega. “So they need something robust and easy to use, something that doesn’t take lots of manipulation and optimization.” In such cases, researchers often turn to master mixes.
Promega’s GoTaq qPCR master mix is based on the company’s proprietary BRYT green dye. “This has a higher fluorescence than others,” says Saldanha, “so it provides earlier detection. It’s great for a lower-expressed target.” She adds that researchers won’t even need to optimize PCR using this mix, unless they are doing something very unusual. “It has been tested with hundreds of templates on a wide range of different instruments to optimize the concentration and performance,” she adds.
Rather than rely on company claims, Promega set up the GoTaq qPCR Challenge to let customers give their opinions of this product. One customer writes: “No amplification of environmental samples was detected with Power SybrGreen. It is worth noting that this assay was not specifically optimized for the GoTaq enzyme (except that extension temperature was lowered to 60º C) and yet the GoTaq enzyme outperformed its competitors.” This customer concludes: “GoTaq is superior to other enzymes for amplification of ‘difficult-to-amplify’ environmental samples. It also provides a much higher sensitivity in our assays…. The GoTaq PCR mix also has an advantage since it does not produce bubbles during pipetting.”
To simplify the PCR process and still get accurate results, researchers can also turn to the high resolution melting (HRM) approach. According to RamaKrishna Badugu, PhD, an applications and technical consultant at Roche Diagnostics, “There are two primary benefits of using the HRM approach: speed and cost effectiveness.” Badugu continues: “This approach does not need expensive probes for detection, but uses a less toxic saturating dye.”
For example, Roche’s LightCycler instrument can be used with the HRM approach to detect methylation of DNA in archival samples, such as formalin-fixed paraffin-embedded (FFPE) tissues. “Methylation is a potential molecular signature for early cancer detection, classification of disease and response to therapy,” says Badugu. “Methylation detection in archival samples results in exposure to a wealth of knowledge that was previously untapped, especially in retrospective clinical research.” For example, a recent study from Roche diagnostics examined promotor methylation in FFPE tissues from people with colon cancer. The HRM approach and LightCycler instrument detected methylated DNA at levels as low as one percent.
Moving ahead with multiplexing
To simultaneously examine targets, PCR needs multiplexing. For instance, Agilent’s MassCode PCR can perform PCR on 30 targets at once. This technology adds mass tags to PCR primers, and then analyzes the amplification products with a mass spectrometer. “Today, this is a custom product being used in a variety of applications—for example, to detect numerous respiratory diseases,” says O’Brien.
Other companies also aim to make PCR easier to multiplex. At Qiagen, for instance, Deutsch points out: “A few years ago, we developed proprietary PCR multiplexing technology that works without any optimization.” Qiagen’s QuantiTect Multiplex PCR kits, for example, can quantify five targets in one PCR tube.
In addition, Qiagen’s Type-it Microsatellite PCR kit—another example for end-point multiplex PCR kits—uses a HotStar Taq Plus DNA polymerase and also multiplexes without optimization. “We developed this for 16-plex,” says Deutsch, “but we have customers doing 30-plex without any limitation.”
Beyond doing more PCR runs at once, researchers can also apply nucleic-acid amplification to more tasks. For instance, Agilent’s DNA Fish ID Ensemble lets researchers use PCR to identify more than 50 species of fish in various food samples. “This can be used to ensure that species claims and fish labeling is accurate,” O’Brien says. Similarly, PCR could be adapted to test products for the presence of genetically modified organisms.
As time and technology move forward, even more applications of PCR will surely develop. This tool grows more useful today for its original purpose of expanding a sample, and it is also takes on new uses, from environmental testing to medical diagnostics and beyond.
About the Author
Mike May, PhD, is a publishing consultant for science and technology based in Minnesota.
This article was published in Bioscience Technology magazine: Vol. 34, No. 8, August, 2010, pp. 1, 10-11.