To summarize, single-base resolution in DNA or RNA remains the goal of investigators now working to establish nanopore sequencing, and several significant intermediate applications are now within reach. These include simple detection of DNA in microscopic volumes, as well as targeted molecular barcodes. Although nanopore analysis is just emerging as a new research tool, the rapidly developing technology has immense promise as an analytical method for measuring and characterizing linear polyions such as nucleic acids. The ability to detect and identify single nucleic acid molecules in solution has numerous applications, but these will be realized only if a stable inorganic nanopore can be developed. We are making significant progress toward this end, and as noted the first such nanopore based on silicon nitride has detected long double-stranded DNA molecules as they pass through. Assuming that a nanopore instrument can be commercially developed, the following applications appear to be feasible:
1. Measurement of nucleic acid concentration, for instance, during PCR amplification.
2. Identifying molecular species in solution. A nanopore in the size range of 4-6 nm diameter will be able to detect and perhaps identify individual soluble protein molecules.
3. Encoded polymers as molecular markers. Akeson et al. (unpublished results) have demonstrated that synthetic nucleic acids can be linked to a cleavable targeting agent and used to identify and quantify analytes in solution. After binding of the targeted marker to the analyte, the linker is cleaved and the nucleic acid marker is determined by its nanopore signal.
4. Hybridized DNA is readily distinguished from single stranded DNA by a nanopore of appropriate size.
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