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McGill Team Develops Single-Molecule Genome Mapping Technology

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NEW YORK (GenomeWeb) – A team of researchers from McGill University in Montreal has developed a technology for single-molecule genome mapping that can be used to resolve long-range genomic information that is often difficult for short-read sequencing technology to do, such as identifying structural variants, resolving repetitive regions, and generating more complete assemblies.

The team described the technology, dubbed convex lens-induced nanoscale templating (CLINT), in the Proceedings of the National Academy of Sciences this month.

Essentially, the technology involves loading a DNA molecule into a nanotemplated structure. Lowering a microscope lens causes the DNA to conform to the underlying structure. In the PNAS study, the researchers created nanochannels around 27 nm in size. DNA molecules are loaded into a solution-based chamber that is separated by two transparent surfaces, in this case a silica coverslip and glass slide. The two substrates are separated by adhesive tape. The lower surface of the chamber contains the embedded nanochannels.

The lens, which is mounted on a nanopositioner device, is gently lowered. At a certain point, the lowering of the lens causes the coiled DNA to stretch out and conform to the nanochannel structures below. The team also demonstrated that DNA would conform to different types of structures, such as nanopits, and different sized nanochannels.

According to Sabrina Leslie, senior author of the study and assistant professor of physics at McGill University, an effect of nanoscale physics causes the DNA to straighten out as the lens is lowered. When DNA is unconstrained, it "prefers to bend and loop around itself," she said. But, when DNA is squeezed into a more constrained space, it develops stiffness, and in a space that is less than 50 nm, the "bending penalty is too high," she said, so it becomes straight.

"What we do is push down on bundles of DNA, lowering the lid of a nanoscale box," Leslie explained. "When we slowly push the DNA down against the bottom of the chamber, they find the [nanochannel] grooves, which they enter and extend along, essentially because they don't want to bend and the grooves provide the perfect opportunity."

While the overall principle of CLINT — stretching out DNA in order to image and map a genome — is very similar to other genomic mapping technology, such as BioNano Genomics' technology, one key difference is how the DNA is loaded.

"In conventional devices, the molecules are pushed into nanoscale orifices from the side with large applied pressure and electric field," Leslie said. In the CLINT device, DNA is loaded from the top, and does not require an electric field or even a large applied force, she said. Instead, as the lens is lowered onto the DNA, the molecules uncoil and stretch to conform to the shape of the nanochannels below. The researchers were able to demonstrate DNA stretching of up to 90 percent.

Next the group demonstrated genomic mapping of the DNA after it had been confined to 50 nm channels. They relied on DNA denaturation using a dye that unbinds from AT-rich regions when they melt, yielding a genomic barcode.

The team showed that after confining the DNA to the nanochannels and heating the flowcell, they were able to generate a barcode-melting pattern. Leslie added that CLINT is also amenable to genomic mapping using restriction enzymes, sequence-specific enzymes, and other biochemical or fluorescent labeling methods, and she said the group is currently working on these applications.

The study suggests that "CLINT-based nanofluidics would be an ideal platform for high-throughput mapping of DNA–protein interactions on extended genomes," the authors concluded.

Michael Rossi, an assistant professor at Emory University who was not affiliated with the study, told In Sequence that the technique could be an important advance. CLINT takes single-molecule genome mapping "to the next step," he said.

Rossi has used BioNano Genomics' Irys technology to map structural variants in multiple myeloma genomes and said that the ability to load DNA from the top would be "a great technical advance" because it wouldn't require the extra complex chemistry and physics steps needed to position and move the DNA into the nanochannels. However, the CLINT technology is far from commercialization, he added.

Leslie said that the researchers are interested in commercializing the technology and are in talks with potential collaborators.

Another potential advantage of CLINT and the ability to top-load DNA is that the amount of force that is required in side-loading devices can often clog or damage the pores, Leslie said.

"Top loading, versus side loading, enables introduction of DNA into nanoconfined dimensions with greater efficiency, control over the rate of analyte introduction by altering the rate of descent of the push-lens, and reduced sensitivity to fabrication defects," the authors concluded.

Leslie added that the technology would be complementary to next-generation sequencing technology, providing longer range genomic information rather than identifying individual bases.

The group is also working on being able to load DNA from individual cells. "That will be the major distinguisher" of the CLINT technique, Leslie said. "We have a nice way of harnessing nanoscale environments to have large pieces of DNA," she said. "So we can take the material from just one cell and look at it as an image or a series of images."

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