By Ben Butkus
In a research paper published this month, scientists from the University of Missouri and molecular biology firm Lucigen have for the first time described a method to amplify DNA transformed into bacterial cells that contain the polymerase needed to drive the reaction.
The technique, dubbed ExCyto PCR, may be a useful tool to increase the throughput, efficiency, and cost of routine nucleic acid amplification, or "dirty PCR," because there is no need to add exogenous enzyme, the researchers said.
As such, Lucigen is currently refining the method in its laboratories in hopes of launching a commercial product – competent E. coli cells designed to overexpress Taq polymerase – by the first quarter of 2011, Lucigen CEO David Mead said this week.
Marjorie Oleksiak and Douglas Crawford, currently researchers at the University of Miami, came up with the idea behind ExCyto PCR while employed at the University of Missouri in the early 2000s.
According to Crawford, they developed the method to male their lives easier when screening, amplifying, and sequencing genes to make microarrays.
"Marjorie came up with the idea of instead of buying or making your own Taq, why not have the bacteria make the Taq?" Crawford told PCR Insider this week. "So you'd make a library in a cell line [expressing] Taq, and since the bacteria already had the Taq polymerase in it, you could screen libraries more readily … to get lots of cDNAs, and to sequence lots of cDNAs to identify them so you could make microarrays."
Since that time, the method has been rendered obsolete for that application due to rapidly evolving sequencing protocols. "People don't screen libraries anymore," Crawford said. However, ExCyto PCR is "still a useful tool," he added, "because it allows you to have higher throughput pretty readily and cheaply."
In a paper published this month in PLoS One, Oleksiak, Crawford, and colleagues at Lucigen described ExCyto PCR for the first time and demonstrated its ability to amplify DNA from different templates, plasmids with different copy numbers, and master mixes left on ice for up to two hours.
They also demonstrated that PCR amplification using ExCyto is comparable to amplification using commercial DNA polymerases from Life Technologies' Invitrogen, Promega, and Stratagene.
"Right now you buy expensive purified Taq polymerase and, depending on what you're doing, you throw it into a dirty [colony] of cells and let heat denature and bust open the cells and do PCR for you," Lucigen's Mead told PCR Insider.
"What was so brilliant about their idea was to manufacture the polymerase in the cell and never purify it," Mead added. "So the same cell that you transformed is the same cell that makes the Taq DNA polymerase so that all you have to do is start thermocycling that cell or collection of cells to do your PCR. You have to add nucleotides and buffer, but you don't have to buy expensive Taq polymerase."
Crawford pointed out that another benefit is that when using the technique, researchers will be able to "just have a vat of nucleotides and primers ready … because you don't have any amplification until you heat up the cells and bust them open and start it. It's almost a hot-start PCR, and we just have a large reservoir of master mix that you can pull off the shelf and use immediately."
Mead said that Middleton, Wis.-based Lucigen, which was founded in 1998, became interested in commercializing the method "because we were so late to the market. We're trying to compete with polymerases … and this might create a new market for competent cells, a product line that would have overexpressed Taq polymerase in it, and would allow you to do your PCR, quick checks on your clones, or what I call dirty PCR, before you move onto something else."
One major drawback to the method thus far is that the first version, based on regular Taq DNA polymerase, failed more than 70 percent of the time for "challenging" templates, such as extremely low-copy-number templates, despite working more than 90 percent of the time for higher copy number templates, Mead said.
"We have several new enzymes that tolerate dirty PCR conditions and low template amounts significantly better than Taq," Mead said. "Once we put them through the gauntlet of tough templates and sort out the best performer we will launch the product. We believe that will be in the first quarter of 2011."
The University of Missouri filed a pair of US patent applications on behalf of Oleksiak and Crawford describing ExCyto PCR earlier this decade, and Lucigen has licensed them from the university. However, this IP might be predated by an emulsion PCR technique described in an earlier patent filed by the UK's Medical Research Council, Crawford and Mead said.
"The people who developed the emulsion PCR in vitro immediately figured out that you could use it to evolve thermostable polymerases," Mead said. "They were making Taq polymerase in the cell and evolving it by mutagenesis. So the patent examiners found that, and although we didn't think this was the same thing, they think there is prior art."
Nevertheless, Lucigen is forging ahead with commercialization of the method and for now leaving any IP disputes to the University of Missouri. Crawford and Mead said that they believe that even if the patents are not awarded, any entity can pursue commercialization now that the ExCyto method has been published.