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Nanopore Researchers Developing Mutation Detection Tech With Single Molecule Sensitivity

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NEW YORK (GenomeWeb) – A research team from the University of Missouri has developed a new methodology that offers the ability to detect mutations at very low frequencies using nanopores.

The approach is based on a technology the team developed called a "nanolock-nanopore" sensor, in which a structure called a nanolock causes DNA moving through a nanopore to undergo a unique type of unzipping, enabling highly sensitive detection of a specific target point mutation.

In a study published this week online in ACS Sensors, the team was able to demonstrate for the first time that its nanolock-nanopores could detect mutations in human tissue — in this case BRAF V600E mutations in thyroid cancer samples.

The researchers anticipate that the approach, once integrated with a miniature, high-throughput device, could enable accurate and PCR-free detection of various disease-causing mutations down not only to levels below 1 percent, but at a single-molecule resolution, regardless of the amount of background DNA.

Liqun Gu, the lead investigator of the project, said this week the nanolock sensor approach could aid efforts in blood-based early cancer detection, and that he and his colleagues are now seeking a grant for that work.

Unlike nanopore sequencing technologies, which are designed to read every nucleotide passing through a pore, the UM team's work has been focused on using nanopores to detect specific target molecules — initially microRNAs, but now DNA point mutations.

According to Gu, current nanopore sequencing approaches don’t have the accuracy required for detection of SNPs at very low frequencies.

"If these devices work perfectly there would be no problem … for detecting SNPs," he said. "But currently, nanopore sequencing accuracy is only 80 to 90 percent."

"With sequencing, you can increase accuracy by increasing sequencing depth, or using machine learning to improve analysis, but at the single molecule level, its still only 90 percent accurate at the most [based on] what we understand from the literature," he added.

Previous work by the Missouri team and others has shown that because nanolocks increase the time needed for a molecule to unzip within a nanopore, they could be used to detect specific target DNA sequences.

But detection based on this longer dehybridization time doesn't provide as precise a readout as would be needed for high-sensitivity mutation detection.

The team's new method relies on their observation that nanolock-bound molecules passing through a nanopore undergo a stepwise dehybridization. As a mutant DNA molecule bound to the nanolock moves through a nanopore, this stepwise unzipping produces a unique electric marker, with which a single DNA molecule of the cancer mutant allele can be unmistakably identified in various backgrounds of the normal wild-type allele, Gu and his colleagues explained in the study.

In the current paper, the team used PCR to amplify both wild type and mutant DNA in order to increase the sample volume. In the future, though, Gu said that if their nanolock approach can be combined with a nanopore device, like the ones developed by Oxford Nanopore, which uses much lower volumes, there would be no need for this amplification step.

Early cancer detection presents an obvious potential application, but the researchers said that the technology also allows quantification, which could be used to monitor levels of cancer mutations in patients being treated with particular drugs or other interventions.

The team is now expanding their analysis to other cancer-associated driver mutations in genes like KRAS and EGFR. They are also planning to develop a system for multiplexing using a barcoding approach they developed for microRNA detection in previous efforts. Their goal is to validate the technology for cancer screening and/or monitoring in collaboration with clinical investigators.

In the ACS Sensors study, the researchers analyzed tumor tissue samples, but Gu said that they are now working on adapting the technology so they can test it for blood-based mutation detection. They have preliminary results already, which they hope to use to apply for additional grant funding.

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