More about the science
We believe that rapid DNA sequencing is a unique application for combining single-molecule electronics and chemical recognition, areas in which our Biodesign lab has significant expertise. The nanotechnology-based sequencing is called nanopore sequencing.
DNA sequencing has progressed from Nobel laureate Fred Sanger’s original method in the early 1970s to more recent, massively parallel approaches that made possible the Human Genome Project and applications in diagnostic and forensic research. The techniques are time consuming, and work with molecular scissors to chop the 3 billion chemical units of DNA that make up the genome into readable bits a few hundred units in length at a time, using a computer to reassemble the full genome sequence.
In concept, nanopore-based DNA sequencing is a bit like sewing, with DNA as the thread, passing through a nanopore like the eye of the needle. Scientists use an electric current to thread the DNA through the nanopore hole.
“One of the most compelling advantages of nanopore sequencing is the prospect of inexpensive sample preparation requiring minimal chemistries or enzyme-dependent amplification,” the authors state. “Thus, the costs of nanopore sequencing, be it by direct strand sequencing or by one of the other methods discussed here, are projected to be far lower than ensemble sequencing by the Sanger method, or any of the recently commercialized massively parallel approaches.”
Nanopore sequencing has the potential to provide a breakthrough in DNA sequencing by reading lengths of DNA up to 50,000 thousand bases in length, and without the need for dyes, sample processing and other materials that contribute to the current costs.
Our variation of the theme, called “sequencing by recognition,” involves using nanostructures to read the electrical current through DNA bases, thereby identifying the sequence (see figure below). In concept, The solution would work somewhat like a supermarket scanner —only shrunk down to the nanoscale— to read genomic DNA at a speed of hundreds to thousands of bases per second. It involves using nanostructures to read the electrical current through DNA bases, thereby identifying the sequence.
We take advantage of a ghostly property of matter, called quantum tunneling. Tunneling permits a particle like an electron to cross a barrier when, according to classical physics, it does not have enough energy to do so.
Our techniques for observing DNA sequences relies on devices known as scanning tunneling- (STM) and atomic force- (ATM) microscopes. These sensitive instruments are used to identify complementary DNA base pairs, evaluating the hydrogen bonds formed between them. Base pairing rules for DNA dictate that the hydrogen bonds work to join up appropriate nucleotide pairs like jigsaw pieces—adenine with thymine and cytosine with guanine.
The scanning tunneling microscope used in our present research features a delicate electrode tip held very close to the DNA sample. When this tip is fitted with a particular nucleotide and brought in contact with its complementary mate—embedded in the substrate, the hydrogen bonds stick the bases together and they attach, like tiny magnets. Sensing chemicals are attached to one electrode and the target you want to sense attached to another one. When the junction spontaneously self-assembles, you get a signal, representing a new way of doing recognition at the atomic scale.
Crucial to the new technique is the fact that the strength of the glue fastening complementary bases differs for A-T and C-G pairs. While two hydrogen bonds hold A-T bases together, C-G pairs use three hydrogen bonds. For this reason, it’s physically harder to break C-G bonds. By measuring the current drop in the electrical circuit formed between the microscope probe and the target base as the hydrogen bonds are gently pulled apart, a positive identification of the base being read can be made. The new method, combines chemical recognition—the hydrogen bonded assembly at the tunnel junction— with the flow of electron tunneling current as the tunneling junction is completed.
In our new study, we made a number of measurements using varying amounts of electrical current through the junction, as the microscope’s electrode is moved away from the substrate and the hydrogen bonds uniting base pairs are slowly stretched and separated. The DNA bases held together by 3 hydrogen bonds, the curves of falling current go on for a long distance. In those held together with two, they go on for less distance.
Electron tunneling is a peculiar property of matter acting over tiny distances at the atomic or subatomic scale. In a classical electric circuit, a gate is either open or closed, permitting or blocking the flow of current. When one starts to get two electrodes so close to one another that they are within a few atomic diameters, then the electrons can actually leak from one electrode to the other, because in quantum mechanics, they’re not confined. These electrons, which violate classical mechanics as they hop across the tiny junction, are said to “tunnel.”
We are now using this method to identify different DNA base pairs, which could serve as the foundation for a new DNA sequencing technology. The tunnel current is there as a readout of how long that molecular pair survived in the junction. But it turns out that it’s an incredibly nice way of identifying which molecular pair it was. Although quantum tunneling seems exotic, the routine leaking of electrons from one atom to another to form a chemical bond is a similar process.
At present, we can determine a composite of 2-10 molecules at a time, with the tunneling currents providing a sort of average stickiness of the hydrogen bonded pairs under examination in the tunnel junction. If significant challenges to reading single molecules through such a technique can be overcome, the method holds the potential for inexpensive DNA sequencing, operating at the breakneck pace of thousands of base pairs per second. This combination of tunneling plus the chemistry can be very powerful.
