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Co-Diagnostics Claims 2.5M-fold Reduction in Non-specific Amplification with New Primer Tech

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This article has been updated from a previous version to correct the name of the vendor that supplied fluorescent probes to Co-Diagnostics.

During PCR, if primers begin to amplify each other it can kick off a propagating reaction, threatening to swamp out PCR signal and leading to false negative results. Hot-start PCR can reduce the problem somewhat, but so-called primer-dimers that form after the initial denaturing step remain a frustrating problem.

DNA Logix and its commercial conduit Co-Diagnostics recently published a paper on a technology that aims to address these problems. The study, which appeared online in The Journal of Molecular Diagnostics last month, describes so-called cooperative primers — short sequences with low melting temperatures that are ordinarily unable to amplify the template.

The short primers won't amplify unless a capture sequence holds them in proximity to the template DNA. This sequence is designed to bind downstream from the primer, with the shortened primer tethered to the capture sequence via a polyethylene glycol linker.

In the study, Co-Diagnostics CTO Brent Satterfield, the sole author of the article, first worked out the theoretical efficiency of the technique ─ one of his particular areas of expertise.

The proof-of-concept experiments compared cooperative primers to primers normally used in PCR. Satterfield measured amplification efficiency on a template of human beta-actin, and used varied probes to measure the impact of primer-dimer and probe selection using plasmodium DNA as a template. He also challenged the technique to differentiate between a SNP conferring drug resistance in tuberculosis DNA versus DNA without the mutation.

Resistance to primer-dimer amplification was shown on gels of PCR products formed after amplifying 60 copies of plasmodium template DNA with or without experimentally added primer-dimers.

With normal primers, a strong signal from 600 spiked-in primer-dimers eclipsed amplification, ultimately resulting in a false-negative PCR. Using hot-start plus normal primers showed little improvement. Cooperative primers, however, did not show a decrease in template amplification ─ even with 600,000 primer-dimers spiked in, the dimers themselves did not amplify.

To take the study a step further, Satterfield attempted to find the absolute limit of spiked-in primer-dimers needed to show some competition with the template using cooperative primers. He found 150 billion primer-dimers were required ─ 2.5 million times more primer-dimers than template ─ to see an eclipse of template amplification.

The author then added a fluorescent probe from Biosearch to the cooperative primers, and was able to find an incorporation position that allowed amplification, as well as a 2.5-fold higher fluorescent signal than normal hybridization probes, even though the capture sequence had a melt temperature below the reaction temperature.

The method also proved able to differentiate SNPs using either labeled capture sequence differentiation or the amplification-refractory mutation system (ARMS) technique.

Satterfield noted in the study that he used a single annealing temperature of 55 ºC and just one type of master mix compatible with single-tube RT-PCR, but asserted that the cooperative primer method should be compatible with a variety of temperatures, as well as different salt or primer concentrations.

Co-Diagnostics recently acquired the cooperative primer technology and related patents from DNA Logix — another of Satterfield's companies — in a stepped-up effort to move DNA Logix technologies to market. The companies also founded Co-Diagnostics HBDC to develop the primers as low-cost molecular diagnostic tools for the developing world.

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