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New Method for Sequencing DNA Shows Promise

Story by
Nancy Joseph

In 2001, Physics Professor Jens Gundlach met a researcher working on nanopore sequencing, a promising new method for sequencing DNA. The research was entirely unrelated to Gundlach’s work as a gravitational physicist, but he was intrigued. 

Jens Gundlach, far right, poses with other Nanopore Physics Lab scientists. The group includes, from left, David Feldman, Liz Manrao, Kyle Langford, Jeney Wierman, Marcus Collins, and Ian Derrington.

Jens Gundlach, far right, poses with other Nanopore Physics Lab scientists. The group includes, from left, David Feldman, Liz Manrao, Kyle Langford, Jeney Wierman, Marcus Collins, and Ian Derrington.

Eight years later, after “digging out a biochemistry book and learning the ropes,” Gundlach now heads his own biophysics lab with funding from the NIH National Human Genome Research Institute.

Why all the excitement about nanopore technology? Unlike traditional DNA sequencing, which Gundlach describes as “expensive and slow,” nanopore sequencing has the potential to be an inexpensive but powerful tool. “If it’s cheap enough, it can be used to do predictive medicine,” Gundlach explains. “It would be a phenomenal medical breakthrough. This is the long-range motivation for us.”

The concept behind nanopore sequencing is simple: A membrane with a tiny opening divides a salt solution into two sections, one connected to a positive electrode, the other to a negative one. Negatively charged DNA slips through the opening to the positive side, modulating the ion current in the opening as it passes through. The modulation varies depending on the type of nucleotide (a subunit of DNA) currently passing through the opening, thus sequencing the DNA. 

Simple idea, difficult to execute. A major challenge is creating an opening of just the right size and shape in the membrane. If the opening is too large or smooth, the DNA will zip through before it can be “read.” If the opening is too small, the DNA will not be able to pass through. Engineered inorganic pores have openings too smooth and uniformly shaped, so Gundlach and other researchers have turned to proteins—pores from nature—for the job. 

Cross-sectional view of an MspA pore with DNA passing through it.

Cross-sectional view of an MspA pore with DNA passing through it. 

“You can buy these proteins,” says Gundlach. “You dump them in there and a couple of minutes later one inserts itself into the bilayer—the membrane—and forms a nanometer-sized hole.” 

Some proteins are preferable to others, since each creates a hole of a different size and shape. Most researchers have used the well-studied pore &alpha-Hemolysin, but recently Gundlach and his group “stumbled across” another protein, Mycobacterium smegmatis porin A (MspA) that seems even better suited to the job. “A researcher at the University of Alabama isolated this pore in totally unrelated tuberculosis research,” says Gundlach. “He didn’t think about it for nanopore sequencing until we contacted him about it.”

When Gundlach’s team first tried MspA, they were disappointed. The negatively charged protein repelled the negatively charged DNA, which meant the DNA would not pass through the opening. The problem was solved by mutating the protein to remove its negative charge. “That’s when our project really took off,” says Gundlach. “Now we’re getting phenomenal results with this thing.”

The next step? The lab is working to control the speed at which DNA travels through the opening. They will soon announce a new approach that involves altering the DNA—making it wider between nucleotides—to slow it down. They also are looking at the possibility of making the walls of the membrane “stickier” to slow down the DNA strands. 

Gundlach hopes that, eventually, an artificial pore can be designed for nanopore sequencing. “It would provide more stability,” he explains. “The pore we’re using, MspA, is really very tough, but the bilayer (membrane) it’s in is not. If the pore were artificial, we wouldn’t need a bilayer at all.”

For now, he’s optimistic about MspA. And he’s pleased that he decided to pursue nanopore research despite being a newcomer to the field. (He continues his gravitational research as well, splitting his time between two labs.) 

“It’s fun,” says Gundlach. “I am learning a lot. Scientists shouldn’t be afraid to take a chance, try things out, change fields. Transitions are possible.”