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      Nanopore-based Fourth-generation DNA Sequencing Technology

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          Abstract

          Nanopore-based sequencers, as the fourth-generation DNA sequencing technology, have the potential to quickly and reliably sequence the entire human genome for less than $1000, and possibly for even less than $100. The single-molecule techniques used by this technology allow us to further study the interaction between DNA and protein, as well as between protein and protein. Nanopore analysis opens a new door to molecular biology investigation at the single-molecule scale. In this article, we have reviewed academic achievements in nanopore technology from the past as well as the latest advances, including both biological and solid-state nanopores, and discussed their recent and potential applications.

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          Most cited references 105

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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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            Solid-state nanopores.

             Cees Dekker (2007)
            The passage of individual molecules through nanosized pores in membranes is central to many processes in biology. Previously, experiments have been restricted to naturally occurring nanopores, but advances in technology now allow artificial solid-state nanopores to be fabricated in insulating membranes. By monitoring ion currents and forces as molecules pass through a solid-state nanopore, it is possible to investigate a wide range of phenomena involving DNA, RNA and proteins. The solid-state nanopore proves to be a surprisingly versatile new single-molecule tool for biophysics and biotechnology.
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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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                Author and article information

                Contributors
                Journal
                Genomics Proteomics Bioinformatics
                Genomics Proteomics Bioinformatics
                Genomics, Proteomics & Bioinformatics
                Elsevier
                1672-0229
                2210-3244
                02 March 2015
                February 2015
                02 March 2015
                : 13
                : 1
                : 4-16
                Affiliations
                [1 ]Chongqing Key Laboratory of Multi-scale Manufacturing Technology, Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing 400714, China
                [2 ]School of Physical Electronics, University of Electronic Science and Technology of China, Chengdu 611731, China
                [3 ]MOE Key Laboratory of Weak-light Nonlinear Photonics, School of Physics, Nankai University, Tianjin 300071, China
                [4 ]University of Chinese Academy of Sciences, Beijing 100049, China
                Author notes
                [* ]Corresponding author. dqwang@ 123456cigit.ac.cn
                [a]

                ORCID: 0000-0002-2513-860X.

                [b]

                ORCID: 0000-0003-0941-2370.

                [c]

                ORCID: 0000-0002-7279-1388.

                [d]

                ORCID: 0000-0002-3151-6769.

                [e]

                ORCID: 0000-0001-9024-0881.

                Article
                S1672-0229(15)00013-3
                10.1016/j.gpb.2015.01.009
                4411503
                25743089
                © 2015 The Authors

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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                single molecule, nanopore, dna sequencing, single base

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