Nanopore Concept

The concept underlying nanopore analysis of DNA is simple: individual nucleic acid molecules are detected as each strand threads through a nanometer-scale opening between two aqueous chambers, typically driven by an applied voltage (Figure 11.1). Two features distinguish nanopore DNA detectors from more conventional DNA detection methods: (i) DNA is sampled directly from bulk-phase solution with no labeling or modification, and (ii) DNA is examined one molecule at a time. Most techniques used to detect DNA amplify the sequence information to a detectable level by using labeling, polymerase chain reaction (PCR), or adhesion to a surface-active detector.34'35 Even techniques used to examine single molecules of DNA such as optical tweezers1 and atomic force microscopy2 typically label DNA or adhere DNA molecules to a surface.

For DNA analysis, the dimensions of a useful nano-pore detector are defined by the size of a single molecule. Single-stranded DNA has a cross section of ~1.2 nm; B-form double-stranded DNA is ~2.2 nm in cross section and each base pair (bp) is ~0.34 nm in length. Thus, an ideal nanopore would be on the order of 1.4 to 3 nm in diameter at its narrowest point to ensure single file presentation of the polymer as it enters and translocates; if single-nucleotide precision is required, then the pore or an embedded detector must be ~0.34 nm thick.

DNA is uniformly negatively charged along its deoxyribose-phosphate backbone in aqueous 1 M salt solutions. This feature makes it straightforward to use an applied voltage to capture and drive DNA through the pore, analogous to the use of an applied field to drive DNA through a conventional sequencing gel or capillary. Voltage is typically applied across the nanoscale pore using standard patch clamp instrumentation. Captured DNA impedes ionic current (Figure 11.2), until either the DNA molecule translocates and exits the pore on the opposite side, or is ejected back into the sampled solution by reversing the polarity of the applied potential. DNA sampling from solution can occur at rates up to thousands of molecules per minute. The data can be used for detailed statistical analyses of the individual molecules and the ensemble.

To date, ionic current impedance has been the only means to detect DNA molecules captured in the pore, but it has been highly informative. Viewed as an extension of the Coulter counter principle,36 we would anticipate current blockades to provide information about molecule size, charge, conformation, and stability. With a nanoscale channel, additional resolution of DNA structure and stability can be expected, because small changes in charge distribution within a channel of these dimensions can give rise to relatively large changes in ionic flux. Such an amplifying transducer allows the detection of very small changes (<0.02kT) in the energy barrier to ionic transport.37'38

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