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QuantiBact Shows How TINA-Modified Primers Increase PCR Efficiency, Robustness

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Danish firm QuantiBact has published a paper describing how its twisted intercalating nucleic acid, or TINA, molecule technology can be used to modify PCR primers to increase the efficiency, specificity, and sensitivity of quantitative and multiplex endpoint PCR assays.

With the publication, the company is hoping to build interest among life science research tool manufacturers and diagnostics companies, with whom QuantiBact is currently negotiating partnerships and potential licensing agreements around the technology, a company official said this week.

In the paper, published this month in PLoS One, the researchers demonstrated that adding ortho-TINA molecules at the 5' position to standard primers in a qPCR experiment enabled 100 percent efficiency in "significantly stressed reaction conditions," including lower primer concentrations and increased annealing temperatures.

The scientists also demonstrated the ability of the o-TINA molecule to increase the sensitivity and specificity of clinical PCR assays in samples spiked with genomic DNA.

Gorm Lisby, QuantiBact's senior vice president and chief science and development officer, told PCR Insider this week that TINA enables more stable primer annealing, using less primer at a higher annealing temperature, making it especially valuable for multiplex PCR "where too much primer will give you unwanted effects such as cross-reaction and false positives and so on."

The ortho-TINA molecule described in the group's recent paper is only one of several different TINA types, Lisby explained. The technology was developed, according to the company's website, from initial work by researchers at Denmark's Hvidovre Hospital and Syddansk University.

In the PLoS One paper, the researchers describe adapting the o-TINA molecule, demonstrated in earlier research for hybridization capture, to PCR.

"The version we are describing is specifically designed … for PCR," Lisby said.

"When you put the TINA molecule at a terminal position in a conventional hybridization probe, it will stabilize the hybridization and increase the melting temperature," he added. "But you cannot do that in a PCR primer because if you put it at the 3' position, you block the polymerase."

"We found [though] that if you put it at the 5' position, you get the same effect — you stabilize the annealing of the primer," he said.

In their study, the researchers set out to test TINA-modified primers in a previously published octaplex end-point PCR assay targeting Escherichia coli. First, however, they tested 5' o-TINA primers using a qPCR assay targeting Neisseria gohorrhoeae. The researchers tweaked reaction conditions to allow the optimal qPCR efficiency with unmodified DNA primers, marking a maximum annealing temperature and minimum primer concentration for this ideal level.

They then incrementally stressed the assay, increasing the annealing temperature and decreasing primer concentration, and tested the efficiency of both unmodified primers and 5' o-TINA-modified primers.

The team reported that unmodified primers allowed qPCR efficiency of 100 percent with primer concentrations 400 nM or less at 66 degrees Celsius or less, while TINA-modified primers showed full efficiency at the same temperature, but all the way down to 200 nM concentration. Further experiments stressing concentration and temperature showed that the TINA-modified primers sustained 100 percent efficiency at both higher temperatures and lower concentrations than unmodified primers, the group reported.

The group also tested whether TINA-modified primers might reduce cross-reactivity in clinical samples with confounding genomic material. To that end, they added 10 or 100 ng of human genomic DNA to the assay, and found that the additions hampered qPCR accuracy for unmodified primers, while the assays using the TINA-modified primers were "unaffected."

The team verified these results using E. coli DNA and observed "similar results," the authors wrote.

According to Lisby, an important feature of the TINA molecule for PCR is that it involves a very strict and simple design rule, making it easy to add to existing assays for a boost in efficiency or robustness. "If you want to take advantage of the TINA technology to increase efficiency of an assay you already designed, you simply add the TINA to the 5' position and you don't need to redesign your primers."

To demonstrate the applicability to multiplex endpoint PCR assays, the researchers tested the impact of the 5' o-TINA modification on an existing octaplex E. coli assay, which was originally described by a Norwegian team in 2007 and which the QuantiBact researchers wrote is "routinely used in clinical microbiology."

The unmodified and TINA-modified primers amplified three targets with equal analytical sensitivity using the published PCR protocol, the group reported. However, when the group reduced the protocol length by 60 minutes, all three amplicons could still be observed using the TINA-modified approach, while the same was not true for the unmodified primers.

As in the qPCR experiment, the TINA-primers also performed well at lower concentrations and higher annealing temperatures, the authors reported. In addition, when human genomic DNA was spiked into the octaplex assay, the TINA-modified primers increased sensitivity and specificity when compared to unmodified primers.

"We were able to lower the primer concentrations and we were able to see signals that were disappearing when we didn't use the TINA addition," Lisby said.

"TINA also seems to allow the primer to accept more complex material because the TINA primers performed better in the genomic DNA background," he added. "This is very interesting for clinical use because if you want to do clinical diagnostics, you would often have a genomic background from whatever patient material you would test, so this would enable you to have a more robust assay."

QuantiBact filed for a patent on its TINA-based PCR approach in 2010.

Lisby said the company is currently in negotiations with companies that make PCR primers for the research community to license the TINA technology. Additionally, he said QuantiBact is in discussions with several potential partners in the diagnostics community who may want to use TINA in their assays.

He declined to identify potential partners or diagnostic applications, but said the PLoS paper's focus on infectious disease might hint at prospects.

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