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Reaction Monitoring Using Mirror-Image DNA Simplifies PCR

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NEW YORK (GenomeWeb) ­– Researchers at Vanderbilt University have discovered that left-handed DNA molecules can be used to monitor temperature changes in PCR reactions, obviating the need for bulky and expensive heat control and detection.

A study published in Analytical Chemistry last month demonstrates that the left-handed enantiomer L-DNA of typical cellular DNA, or D-DNA, does not interact with PCR reactions. However, L-DNA does have thermal and chemical characteristics identical to D-DNA, enabling what is essentially a parallel in-tube reaction to monitor melt.

Typical PCR requires temperature sensing of the reaction, or a proxy region nearby, to indicate that a given cycle temperature has been reached and it is time to switch to the next cycle temperature. But by mimicking the reaction with L-DNA that is fluorescently tagged, much of the heaviest and finickiest components of a thermal cycler can be eliminated.

"We envision long-term this might be made in the scale of like a FedEx tracking device," Frederick Haselton, a corresponding author on the study and professor of biomedical engineering at Vanderbilt, said in an interview. A small size is enabled by the fact that the device does not require thermal insulation or regulatory elements, he said.

L-DNA was first synthesized in 1984 with the hopes that it could bind to promoter regions and silence transcription, Nicholas Adams, a biomedical engineer at Vanderbilt and first author on the study, said. At the time, investigators were intrigued by the fact that L-DNA is not degraded by cellular enzymes and is fairly easy to synthesize. However, they were ultimately disappointed because it does not bind or interact with natural DNA. "It's like trying to get a right-handed glove onto a left hand," Adams said.

There were a smattering of studies in the 1980's and 1990's and then L-DNA research tapered off, he said, only to be revived in the early 2000's with L-DNA being used as a biologically inactive probe, a nuclease resistant aptamer, and a non-cross-reactive microarray tag.

Adams and Haselton had been working on a Bill and Melinda Gates Foundation-funded project developing simpler instruments for low-cost diagnostics for about three years when the brainstorm struck to use L-DNA.

"Like many stories in science, it started in a car," Adams said. The two were driving back from a meeting with a collaborator and were discussing PCR instrument design, trying to come up with more direct ways to measure temperature.

"Measurements are taken outside of the tube, and because of that there are calibrations and algorithms used to predict the temperature inside the tube — we were trying to get around that," he said. They discussed probes and other systems, but then stumbled on the idea: "Why monitor temperature at all, why not just monitor the molecular events?" Adams said.

The obvious hurdle was finding a way to monitor primers binding to targets when targets start out at such low concentrations, he said, as well as finding a way to monitor melting when the molecules don't have probes on them.

"In the previous week, I read a paper where somebody had been using L-DNA for monitoring cell processes, so I said, 'What if we just mimic the reaction,'" Adams said. The method they devised uses a primer mimic as well as mimics of the two strands of the product.

However, they first had the problem of figuring out where to get L-DNA. "It's funny to look back, but at the time I didn't even know if you could buy this stuff, and I'm not a synthetic chemist so I couldn't make it myself," Adams said. The lab ultimately found two suppliers, Bio-Synthesis and Biomers, that can synthesize labeled L-DNA.

The researchers also needed to confirm that spiked-in L-DNA did not interfere with a PCR reaction, and that it faithfully represented the changes in a reaction due to variations in salt concentrations. Their experiments showed both to be the case.

In a prototype instrument using the L-DNA method the researchers suspended PCR tubes from Cepheid in front of a heat gun that blasts heated air, much like a hair dryer, to drive the reaction. "It could be run with Peltier heater and coolers, but we went with simple first," Haselton said.

The temperature of the heat gun is not regulated well, but that only underscores the beauty of the method, Adams said. "We don't need to control heat precisely — because we are directly monitoring interactions using the L-DNAs, and [because] they indicate what is going on in the DNAs, you can get away with unstable thermal conditions and still perform PCR very precisely and accurately," he said.

The tagged L-DNA in the tube emitted fluorescent signal that was detected with an ESElog optical reader from Qiagen, but Haselton suggested that a future device will have a custom-built reader that should provide faster optical sampling rates as well as measurements even more faithful to the timing of the actual molecular events.

Importantly, unnatural L-DNA has never been discovered in cells, adding a level of specificity to the method. "We don't know why evolution turned out to be right-handed but there was clearly a checkpoint somewhere way back in the universe where right-handed won out over left-handed," Haselton said.

The team has been collaborating with a local company called BioVentures, and recently created its own start-up, Mirror Molecular.

The group has also filed a patent application on the method and has had early licensing discussions with several firms in the diagnostics space. 

Adams noted that large firms that have been interested seem to be too far down development pathways to add this method and start a new regulatory trajectory. But the researchers are now considering reaching out to small- and medium-size diagnostics manufacturers.

Future directions for the research include further development of the instrument and testing it using clinical samples. The goal is to perform PCR without sample prep, and the researchers are working with the Vanderbilt medical center to obtain urine samples to detect urinary tract infection pathogens such as E. coli as well as sexually transmitted disease pathogens. 

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