BIOPHYSICS OF DNA

COLLABORATOR: Prof. Dan Branton

DESCRIPTION

The goal of this project is to develop a revolutionary electronic method of very rapidly sequencing DNA using only a single molecule. The basic idea is to create a liquid cell which is separated into two compartments by a thin insulating membrane which contains a single hole or pore whose diameter is just slightly greater than the diameter of a single stranded polymer of DNA (about 20 Angstroms). The liquid in the cell is a highly conducting salt solution. If a voltage is applied across the two compartments of the cell an ionic current flows through the cell that is limited by the size of the constricting pore. Because the pore is extremely small the entire voltage drop will appear across it, and consequently a large electric field will exist within the nanopore. (Pores of a few nanometer diameter will be called nanopores from here on.)

Single stranded DNA polymeric molecules are negatively charged (about 1 charge every 3.5 Angstroms) in a salt solution, and as a result, if the tail of one of these molecules on the positive side of the cell comes close to the pore it will be drawn in and translocated from one side of the membrane to the other. When this happens, the pore will be blocked and one might expect the transmembrane resistance to increase, with a corresponding drop in the ionic current that passes from one side of the cell to the other.

If it were true that different bases on the DNA polymer blocked the pore differently then it would follow that a different current would flow depending on which base is currently in the pore. The hope is that as this spaghetti-like polymer passes from one side of the cell to the other a modulated current will flow which can be read electronically for its genetic code like a ticker tape.

In addition to the obvious practical utility of such a device, the project involves understanding many areas of physics that contribute to the desired effects. For example, a microscopic understanding of current flow in the pore, and the mechanical speed at which the polymers pass through the pore are not well understood at all. In addition, the fluctuations in these quantities will play a major role in determining whether this strategy for gene sequencing can succeed. Whether or not it does succeed, the knowledge gained will be an invaluable part of our understanding of atomic scale biological processes.

One experimental approach has been two pronged, using membranes and nanopores fabricated using the methods of molecular biology and modern methods from the semiconductor world of nanotechnology. To date we have succeeded in detecting signals fron DNA passed through lipid membranes containing a single alpha hemolysin-based nanopore. It has proved possible to establish that there is a significant difference between artificially prepared DNA molecules consisting of all A or all C bases. Sufficient time resolution for atomic scale sequencing has not yet been achieved.

On the solid-state nanopore front, we have recently succeeded in preparing 5 nanometer pores in silicon nitride using a newly invented single atom sensitive, feedback controlled atomic sputtering system used in conjunction with modern Focused Ion Beam facility. Electrical measurements suggest these pores will soon be suitable for biological applications.