Real-time PCR gets faster and smaller, and decades-old gel electrophoresis faces new competition with the advent of microfluidics
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For expression screening, Agilent’s 5100 ALP was used to perform post-purification analysis via affinity tag on enzymes produced in a baculovirus system. (Image: Paul Hawtin, AstraZeneca)
As in other areas of drug discovery, new microfluidic technologies are helping researchers do more with less. Researchers at Johnson & Johnson Pharmaceutical Research and Development (J&JPRD), Spring House, Pa., have been using some of the latest microfluidic technologies to increase their rate of polymerase chain reaction (PCR) analysis from three or four to nearly 9,000 in three hours.
“We can analyze many samples and it doesn’t cost us so much as before because the [reaction volumes] are basically reduced to essentially nanoliters. So, we have a volume reduction of at least a thousand-fold,” says Serge Ilyin, PhD, bioinformatics group leader, J&JPRD. “That makes things affordable, and because things are affordable, we can now run more assays and know much more about our compounds. Going forward, we can reduce our risks because we can perform many additional assessments at low cost.”
Paul Hawtin, PhD, senior research scientist, global sciences and information, AstraZeneca, Alderly Park, UK, is using the latest-generation liquid chromatography technology to speed quantitative and qualitative analysis of proteins. Hawtin and colleagues are beta-testing the 5100 Automated Lab-on-a-chip Platform (ALP) from Agilent Technologies Inc., Palo Alto, Calif. “This is really our lab’s first in-depth look into microfluidics and actually using a microfluidics instrument, and it’s been a very positive experience so far with the beta test of this instrument. It opens up our scope to the types of things that these instruments can do, and we obviously may be looking at other things in the future,” Hawtin says of the 5100 ALP, which was launched in November.
Ilyin has been beta-testing the OpenArray NT Cycler, a real-time PCR system from BioTrove Inc., Woburn, Mass., which will be available in the spring. Ilyin’s lab got the first system in the United States and has been using it to identify signatures that they were previously unable to see using other genomic tools. The OpenArray reduces reaction volume to 33 nL, so in a small glass tube, researchers can conduct 3,000 individual PCR reactions, Ilyin says. In addition, array plates can be loaded into the device three at a time, allowing for simultaneous study of more than 9,000 samples. “Ultra-high-throughput platforms such as BioTrove enable us to substitute microarrays with real-time PCR approaches,” he says.
Typically, researchers would expend a great deal of time and energy conducting microarray experiments to select a group of genes that would then be confirmed using real-time PCR, a process that could often take more than a month, Ilyin says. “Using BioTrove’s technology, we can essentially start with PCR experiments so they can get an answer on the same day, as opposed to waiting a month or more, and it gives us high-quality data.”
Ilyin says he sees other applications for the system in toxicogenomics and pharmacogenomics, so while they are now looking at expression, they would like to look at things such as specific point mutations, something that is essential for targeted therapy approaches. They are currently looking for one sample across 3,000 genes, but in the near future they plan to set up a configuration that will allow them to look at 3,000 different samples against maybe one or two factors.
“In this case, we would be able to use this platform for lead selection and lead optimization. In other words, it will be a very powerful screening platform,” he says. Ilyin believes the technology’s simplicity and low cost will “transform” the role of bioinformatics and genomics in the industry. “It can be used across different segments of the business, so things which before were restricted to a few highly specialized labs will be used in many, many different areas. That way, it would basically increase the impact of genomics or whole-genome sequencing on day-to-day operations.”
In October, Applied Biosystems Inc., Foster City, Calif., introduced what it calls the first real-time PCR system offered in a traditional 96-well format. The 7900HT Fast Real-Time PCR System reduces thermal cycling times from two hours to 35 minutes when compared to standard real-time PCR systems, the company says.
The 7900HT allows researchers to run fast PCR in a standard 96-well format, as well as giving them the ability to interchange thermal cycling blocks of different formats, such as a 384-well block and the TaqMan Low Density Array block, which provides built-in flexibility.
Raghu Viswanath, an associate scientist at the Altana Research Institute, Waltham, Mass., says he is happy with the performance of the TaqMan Low Density Array. “It reduces the size of your reaction tremendously, it improves the efficiency of your reaction, and at the same time it reduces errors while increasing your reproducibility and giving you the best possible sensitivity,” says Viswanath, who works on identifying targets for oncology, inflammation, respiratory diseases, and other disorders for the company, which is based in Germany. “I think certainly it has increased the speed with which we do things, it has increased the number of samples that we have processed in a given time.”
Another technological advance in microfluidics has come in the field of liquid chromatography. Agilent’s 5100 ALP can provide unattended analysis of thousands of DNA and protein samples daily and is designed to replace SDS-polyacrylamide gel electrophoresis (SDS-PAGE), one of the oldest and most widely used analytical techniques. SDS-PAGE is also a technology that has not changed in 40 years, says Derek Barratt, PhD, team leader in protein science at AstraZeneca and a colleague of Hawtin.
The 5100 ALP automates the SDS-PAGE workflow of sample handling and analysis, lowering per-sample analysis time by a factor of 10 and allowing unattended analysis of up to 3,840 samples per run, Agilent says. Unlike the variable data of SDS-PAGE, the 5100 ALP’s automation and use of internal reference standards provides reproducible and comparable data that can be archived and mined. Two assays consisting of chip and reagents are initially available. The first is the DNA 1000 HT-4 kit for qualitative and quantitative verification of PCR and multiplex PCR samples, or for screening of double-stranded DNA fragments. The second is the Protein 200 HT-2 kit for sizing and quantification of proteins during purification or protein-expression experiments.
Hawtin and Barrett have been beta testing the 5100 ALP for several months after it was introduced to deal with some of their laboratories’ high-throughput needs. “We have our traditional routes of protein production, but we also have new methodologies now,” Hawtin says. “We are trying to produce protein more quickly and are also looking at more parameters of protein production in a single experiment. The one thing that we were lacking in that area was a good, robust method for analyzing the protein in both a qualitative and quantitative fashion.” Such analysis was traditionally performed using SDS-PAGE, which does the job but is also a slow, labor-intensive process.
Hawtin says the 5100 ALP is unique in its ability to automate the process to such an extent and in the number of samples it can test. The researchers are now able to test up to 400 or 500 different conditions in a single experiment and then analyze the protein that comes out of the back end. They can do this in an automated manner in which the system is left to run overnight. “We return in the morning, analyze it, and interpret the data to see which are the best conditions that we can pursue,” Hawtin says.
Filed Under: Drug Discovery