The Human Genome Project was a scientific breakthrough that harnessed an international consortium of experts and required 13 years to complete. It was a rewarding process, but also a grueling one that didn't initially lend itself to efficiency. But with new technology, researchers are attempting to cut sequencing time exponentially—not to years, but to minutes. Among those involved in the ambitious research is Associate Professor of Physics and Astronomy Marija Drndic. Along with her fellow researchers Christopher Merchant and Meni Wanunu—both post-docs in the Department of Physics and Astronomy—Drndic was exploring the electronic properties of graphene, a two-dimensional sheet of carbon only one atom thick. When she came upon literature suggesting that DNA could be detected and sequenced by threading it through nanopores—holes 2-3 x 10-9 meters in diameter—she saw a connection to the work she was doing in the lab. Given the familiarity she and her fellow researchers already had with graphene's properties, she jumped at the chance to explore new possibilities in regards to DNA threading.
Using their prior expertise, Drndic’s group soon detected microRNAs using very thin silicon nitride nanopores, work that was featured in the November 2010 issue of Nature Nanotechnology. MicroRNAs were first identified in 1993 and function to dampen the expression of certain proteins by binding to the messenger RNAs. MicroRNAs are very difficult to detect because they are extremely small molecules and are present in only a few copies per cell. But with nanopores, this limited starting material does not pose a problem. The pore is cut into a piece of silicon nitride that divides two chambers filled with electrolyte solution. When voltage is applied, the current of ions moving through the pore is measured. Similarly, when a biomolecule such as DNA or microRNA is passing through the nanopore, it blocks the flow of ions and can thus be detected as a drop in current.
"Because graphene is so thin—thinner than the distance between adjacent nucleotides in DNA’s double helix—it is ideal for sequencing DNA." - Marija Drndic
In a subsequent study, published in the Journal of the American Chemical Society, Drndic’s team sought to use nanopores to identify and quantify epigenetic changes (modifications in the DNA molecule that do not involve alterations in its nucleotide sequence). These modifications play important roles in such processes as embryonic development, aging and tumor formation. However, it has been difficult to distinguish how much DNA in a given cell is subject to modification. Drndic’s team took advantage of the fact that each modification affects the mechanical properties of DNA differently when the strand is passed through a nanopore.
Drndic explains that “the electronic properties of graphene nanostructures depend on their geometry.” In pursuing these properties in 2008, Drndic was interested in making ribbons and other shapes out of graphene, and poking holes in it was the first step in this process. Because graphene is so thin—thinner than the distance between adjacent nucleotides in DNA’s double helix—it is ideal for sequencing DNA. “Graphene is conducting, while silicon nitride is insulating, but both materials may be used towards DNA sequencing.” The team is also developing a platform based on nanoelectrodes positioned near silicon nitride pores, and is hoping to detect the unique electronic signature of each nucleotide—A, T, C, and G—as it passes through the nanopore.
Current methods of DNA sequencing rely on optical measurement of these four bases, and require amplification of the DNA to provide enough starting material. Moreover, these methods can read only a few hundred nucleotides at a time, so the DNA must be chopped into small pieces prior to sequencing. Sequencing DNA as it is threaded through nanopores has the potential to alleviate most of these bottlenecks. Since nucleotide discrimination is electronic, and not optical, no amplification is needed, and in theory an entire chromosome could be threaded through in one piece.
The Drndic lab’s work is featured in an article released by the National Human Genome Research Institute titled “The Road to the $1,000 Genome,” as well as in the March 10, 2011 issue of The Economist in an article titled, “Towards the 15-minute Genome.”
Drndic is eager to use the new technology to help educate undergraduates in the lab. “Physics students at Penn are phenomenal in their work ethic, and their ambition,” she says. “It’s a great pool of students and they are eager to explore new techniques—they are a great resource for us.”
