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The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters. Voltage-Driven DNA Translocations through a Nanopore We measure current blockade and time distributions for single-stranded DNA polymers during voltagedriven translocations through a single a-hemolysin pore. We use these data to determine the velocity of the polymers in the pore. Our measurements imply that, while polymers longer than the pore are translocated at a constant speed, the velocity of shorter polymers increases with decreasing length. This velocity is nonlinear with the applied field. Based on this data, we estimate the effective diffusion coefficient and the energy penalty for extending a molecule into the pore.

Solid-state nanopores are sensors capable of analyzing individual unlabelled DNA molecules in solution. While the critical information obtained from nanopores (e.g., DNA sequence) is the signal collected during DNA translocation, the throughput of the method is determined by the rate at which molecules arrive and thread into the pores. Here we study the process of DNA capture into nanofabricated silicon nitride pores of molecular dimensions. For fixed analyte concentrations we find an increase in capture rate as the DNA length increases from 800 to 8,000 basepairs, a length-independent capture rate for longer molecules, and increasing capture rates when ionic gradients are established across the pore. In addition, we show that application of a 20-fold salt gradient enables detection of picomolar DNA concentrations at high throughput. The salt gradients enhance the electric field, focusing more molecules into the pore, thereby advancing the possibility of analyzing unamplified DNA samples using nanopores.

A variety of different DNA polymers were electrophoretically driven through the nanopore of an ␣-hemolysin channel in a lipid bilayer. Single-channel recording of the translocation duration and current flow during traversal of individual polynucleotides yielded a unique pattern of events for each of the several polymers tested. Statistical data derived from this pattern of events demonstrate that in several cases a nanopore can distinguish between polynucleotides of similar length and composition that differ only in sequence. Studies of temperature effects on the translocation process show that translocation duration scales as ϳT ؊2 . A strong correlation exists between the temperature dependence of the event characteristics and the tendency of some polymers to form secondary structure. Because nanopores can rapidly discriminate and characterize unlabeled DNA molecules at low copy number, refinements of the experimental approach demonstrated here could eventually provide a low-cost high-throughput method of analyzing DNA polynucleotides.T he discovery that a voltage gradient can drive single-stranded RNA or DNA molecules through a 2-nm transmembrane channel, or nanopore, has opened up the possibility of detecting and characterizing unlabeled polynucleotide molecules at low copy number by using single-channel recording techniques. Because an extended molecule of DNA or RNA can occupy, and thus block, much of an otherwise open aqueous channel, the passage of a single polynucleotide can be monitored by recording the translocation duration and blockade current (magnitude of the reduced ionic flow through the pore) (1). Studies with RNAs of differing base composition have begun to suggest how nanopores could be used to discriminate between different nucleic acid polymers (2).We now extend these observations and provide evidence that each of several different DNA polymers can be identified by a unique pattern in ''event diagrams,'' which are plots of translocation duration vs. blockade current for an ensemble of events. Patterns for a given polymer can be characterized uniquely by three statistical parameters representing the most probable translocation current, I P , the most probable translocation duration, t P , and the characteristic dispersion of values for individual translocation durations, T . Because each type of polynucleotide we use gives rise to specific values of these three parameters, these parameters' values can be used to discriminate rapidly between different types of polynucleotides in a mixed sample. Temperature markedly affects these parameters in two ways. The translocation duration scales as T Ϫ2 for all polymers tested, where T is temperature in°C. The other two parameters exhibit a strong temperature dependence for only those polymers that are known to have stable secondary structure. Materials and MethodsSingle channels were formed in a horizontal bilayer of diphytanoyl phosphatidylcholine by using the protein ␣-hemolysin from Staphylococcus aureus. The horizontal bilayer was formed across a ...