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Review
. 2008 Oct;26(10):1146-53.
doi: 10.1038/nbt.1495.

The potential and challenges of nanopore sequencing

Daniel Branton  1 , David W Deamer, Andre Marziali, Hagan Bayley, Steven A Benner, Thomas Butler, Massimiliano Di Ventra, Slaven Garaj, Andrew Hibbs, Xiaohua Huang, Stevan B Jovanovich, Predrag S Krstic, Stuart Lindsay, Xinsheng Sean Ling, Carlos H Mastrangelo, Amit Meller, John S Oliver, Yuriy V Pershin, J Michael Ramsey, Robert Riehn, Gautam V Soni, Vincent Tabard-Cossa, Meni Wanunu, Matthew Wiggin, Jeffery A Schloss
Affiliations
Review

The potential and challenges of nanopore sequencing

Daniel Branton et al. Nat Biotechnol. 2008 Oct.

Abstract

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 VSports手机版. 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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"V体育ios版" Figures

Figure 1
Figure 1
Approaches to nanopore sequencing. (a) Strand-sequencing using ionic current blockage. A typical trace of the ionic current amplitude (left) through an α-hemolysin pore clearly differentiates between an open pore (top right) and one blocked by a strand of DNA (bottom right) but cannot distinguish between the ~12 nucleotides that simultaneously block the narrow transmembrane channel domain (red bracket). (b) Exonuclease-sequencing by modulation of the ionic current. An exonuclease (pale blue) attached to the top of an α-hemolysin pore through a genetically encoded (deep blue), or chemical, linker sequentially cleaves dNMPs (gold) off the end of a DNA strand (in this case, one strand of a double-stranded DNA). A dNMP's identity (A, T, G or C) is determined by the level of the current blockade it causes when driven into an aminocyclodextrin adaptor (red) lodged within the pore. After a few milliseconds, the dNMP is released and exits on the opposite side of the bilayer. (c) Nanopore sequencing using synthetic DNA and optical readout. Each nucleotide in the target DNA that is to be sequenced is first converted into a longer DNA strand composed of pairs of two different code-units (colored orange and blue for illustration); each code-unit is a 12-base-long oligomer. After hybridizing the converted DNA with molecular beacons that are complementary to the code units, these beacons are stripped off using a nanopore. The sequence of the original DNA is read by detecting the discrete short-lived photon-bursts as each oligo is stripped. (d) Strand-sequencing using transverse electron currents. DNA is driven through a nanopore functionalized with embedded emitter and collector tunneling probes (orange) and a back gate (black). The amplitude of the tunneling currents that traverse through the nucleotides is expected to differentiate each nucleobase as the DNA is electrophoretically driven through the pore (arrow).
Figure 2
Figure 2
A nanopore reader with chemically functionalized probes. As a strand of DNA emerges from a nanopore, a `phosphate grabber' on one functionalized electrode and a `base reader' on the other electrode form hydrogen bonds (light blue ovals) to complete a transverse electrical circuit through each nucleotide as it is translocated through the nanopore.
See this image and copyright information in PMC

References

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