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      Electrostatic Focusing of Unlabeled DNA into Nanoscale Pores using a Salt Gradient

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          Abstract

          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.

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          Most cited references32

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          The potential and challenges of nanopore sequencing.

          A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of 'third generation' instruments that will sequence a diploid mammalian genome for approximately $1,000 in approximately 24 h.
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            Continuous base identification for single-molecule nanopore DNA sequencing.

            A single-molecule method for sequencing DNA that does not require fluorescent labelling could reduce costs and increase sequencing speeds. An exonuclease enzyme might be used to cleave individual nucleotide molecules from the DNA, and when coupled to an appropriate detection system, these nucleotides could be identified in the correct order. Here, we show that a protein nanopore with a covalently attached adapter molecule can continuously identify unlabelled nucleoside 5'-monophosphate molecules with accuracies averaging 99.8%. Methylated cytosine can also be distinguished from the four standard DNA bases: guanine, adenine, thymine and cytosine. The operating conditions are compatible with the exonuclease, and the kinetic data show that the nucleotides have a high probability of translocation through the nanopore and, therefore, of not being registered twice. This highly accurate tool is suitable for integration into a system for sequencing nucleic acids and for analysing epigenetic modifications.
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              DNA molecules and configurations in a solid-state nanopore microscope.

              A nanometre-scale pore in a solid-state membrane provides a new way of electronically probing the structure of single linear polymers, including those of biological interest in their native environments. Previous work with biological protein pores wide enough to let through and sense single-stranded DNA molecules demonstrates the power of using nanopores, but many future tasks and applications call for a robust solid-state pore whose nanometre-scale dimensions and properties may be selected, as one selects the lenses of a microscope. Here we demonstrate a solid-state nanopore microscope capable of observing individual molecules of double-stranded DNA and their folding behaviour. We discuss extensions of the nanopore microscope concept to alternative probing mechanisms and applications, including the study of molecular structure and sequencing.
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                Author and article information

                Journal
                101283273
                34218
                Nat Nanotechnol
                Nature nanotechnology
                1748-3387
                1748-3395
                30 December 2009
                20 December 2009
                February 2010
                1 August 2010
                : 5
                : 2
                : 160-165
                Affiliations
                [1 ]Department of Biomedical Engineering and Department of Physics, Boston University, Boston, MA 02215
                [2 ]Department of Physics, Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat-Gan 52900 Israel
                [3 ]Department of Physics, New York University, New York, NY 10003
                Author notes
                [* ]Corresponding author. ameller@ 123456bu.edu
                Article
                nihpa159157
                10.1038/nnano.2009.379
                2849735
                20023645
                f5e23fae-4a3c-4daa-b913-56f0e761c576
                Funding
                Funded by: National Human Genome Research Institute : NHGRI
                Award ID: R01 HG004128-03 ||HG
                Funded by: National Human Genome Research Institute : NHGRI
                Award ID: R01 HG004128-02 ||HG
                Categories
                Article

                Nanotechnology
                Nanotechnology

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