Nanotechnology, DNA sequencing, and personalized medicine

DNA through a nanopore in graphene
Credit: Lab of Jene Golovchenko, Harvard University

Artist’s conception of a nanopore drilled into a layer of graphene to speed up DNA sequencing.

One of the greatest promises of near-term nanotechnoloogy is cheaper DNA sequencing to speed the development of personalized medicine. There are not only genetic differences between different patients, but also genetic differences between, for example, different cancers of the same organ diagnosed in different patients, or even from different locations in the same patient, that can greatly affect the success of a therapy. Nanopore sensors are among the promising new third-generation DNA sequencing technologies being developed to make inexpensive whole genome sequencing a reality. A review of the potential of this emerging nanotechnology was published recently in Nature Nanotechnology [abstract]. The full text of the review “Nanopore sensors for nucleic acid analysis” has been made available by the authors for down-loading. Nanopores and other third generation sequencing technologies sequence single molecules of DNA in real time. Single molecules of DNA are pulled through a nanopore of some type and changes in the ionic current, dependent on whether an A, G, C, or T nucleotide is passing through the pore, are recorded. The review discusses the different types of nanopore that have been tried, both biological and solid-state, and the challenges encountered, such as reducing the speed at which the DNA molecule transits the nanopore, and improving sensitivity.

Research done by scientists at Harvard and MIT and published in Nature [abstract, free authors’ manuscript deposited in PubMedCentral] showed that a graphene sheet one or two atomic layers thick could form an electrode separating two liquid reservoirs so that current from ions passing through a nanopore in the graphene sheet could be measured, and the current blockade seen when DNA molecules passed through the pore indicated it should be possible to resolve individual nucleotides with an insulating membrane this thin. From a Harvard Gazette article by Michael Rutter “Graphene may help speed up DNA sequencing“:

… By drilling a tiny pore just a few nanometers in diameter, called a nanopore, in the graphene membrane, the researchers were able to measure exchange of ions through the pore and demonstrate that a long DNA molecule can be pulled through the graphene nanopore just as a thread is pulled through the eye of a needle.

“By measuring the flow of ions passing through a nanopore drilled in graphene we have demonstrated that the thickness of graphene immersed in liquid is less then 1 nm thick, or many times thinner than the very thin membrane which separates a single animal or human cell from its surrounding environment,” says lead author Slaven Garaj, a physics research associate at Harvard. “This makes graphene the thinnest membrane able to separate two liquid compartments from each other. The thickness of the membrane was determined by its interaction with water molecules and ions.” …

“Although the membrane prevents ions and water from flowing through it, the graphene membrane can attract different ions and other chemicals to its two atomically close surfaces. This affects graphene’s electrical conductivity and could be used for chemical sensing,” says co-author Jene Golovchenko, the Rumford Professor of Physics and Gordon McKay Professor of Applied Physics at Harvard, whose pioneering work started the field of artificial nanopores in solid-state membranes. “I believe the atomic thickness of the graphene makes it a novel electrical device that will offer new insights into the physics of surface processes and lead to a wide range of practical application, including chemical sensing and detection of single molecules.” …

When the researchers added long DNA chains in the liquid, they were electrically pulled one by one through the graphene nanopore. As the DNA molecule threaded the nanopore, it blocked the flow of ions, resulting in a characteristic electrical signal that reflects the size and conformation of the DNA molecule. …

As a DNA chain passes through the nanopore, the nucleobases, which are the letters of the genetic code, can be identified. But a nanopore in graphene is the first nanopore short enough to distinguish between two closely neighboring nucleobases.…

More recently another group at Harvard has integrated nanowire field-effect transistors with a solid-state nanopore to achieve rapid, sensitive detection of the very small currents created as DNA molecules zip through the nanopore. From a Harvard Gazette story by Peter Reuell “Reading life’s building blocks“:

Scientists are one step closer to a revolution in DNA sequencing, following the development in a Harvard lab of a tiny device designed to read the minute electrical changes produced when DNA strands are passed through tiny holes — called nanopores — in an electrically charged membrane.

As described in Nature Nanotechnology [abstract, free full text provided by authors] on Dec. 11, a research team led by Charles Lieber, the Mark Hyman Jr. Professor of Chemistry [and also winner of the 2001 Feynman Prize in Nanotechnology-Experimental], have succeeded for the first time in creating an integrated nanopore detector, a development that opens the door to the creation of devices that could use arrays of millions of the microscopic holes to sequence DNA quickly and cheaply.

First described more than 15 years ago, nanopore sequencing measures subtle electrical current changes produced as the four base molecules that make up DNA pass through the pore. By reading those changes, researchers can effectively sequence DNA.

But reading those subtle changes in current is far from easy. A series of challenges — from how to record the tiny changes in current to how to scale up the sequencing process — meant the process has never been possible on a large scale. Lieber and his team, however, believe they have found a unified solution to most of those problems.

“Until we developed our detector, there was no way to locally measure the changes in current,” Lieber said. “Our method is ideal because it is extremely localized. We can use all the existing work that has been done on nanopores, but with a local detector we’re one step closer to completely revolutionizing sequencing.”

The detector developed by Lieber and his team grew out of earlier work on nanowires. Using the ultra-thin wires as a nanoscale transistor, they are able to measure the changes in current more locally and accurately than ever before.

“The nanowire transistor measures the electrical potential change at the pore and effectively amplifies the signal,” Lieber said. “In addition to a larger signal, that allows us to read things much more quickly. That’s important because DNA is so large [that] the throughput for any sequencing method needs to be high. In principle, this detector can work at gigahertz frequencies.”

The highly localized measurement also opens the door to parallel sequencing, which uses arrays of millions of pores to speed the sequencing process dramatically.

In addition to the potential for greatly improving the speed of sequencing, the new detector holds the promise of dramatically reducing the cost of DNA sequencing, said Ping Xie, an associate of the Department of Chemistry and Chemical Biology and co-author of the paper describing the research. …

“Right now, we are limited in our ability to perform DNA sequencing,” Xie said. “Current sequencing technology is where computers were in the ’50s and ’60s. It requires a lot of equipment and is very expensive. But just 50 years later, computers are everywhere, even in greeting cards. Our detector opens the door to doing a blood draw and immediately knowing what a patient is infected with, and very quickly making treatment decisions.”

Rapid, inexpensive DNA sequencing and other nanotechnology-based innovations in drug-delivery and tissue regeneration may transform health care in the coming decade.
—James Lewis

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