The potential and challenges of nanopore sequencing

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. 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 ~$1,000 in ~24 h.

Disentangling Steric and Electrostatic Factors in Nanoscale Transport Through Confined Space

“…In nanopore analytics, single molecules are detected via the passage-induced changes in ionic current. [2][3][4][5][6][7] The approach can be implemented with protein 4,[8][9][10] and solid-state nanopores [10][11][12][13][14][15][16] and has gained popularity 17 due to its simplicity, the wide range of accessible analytes, 2,13,[18][19][20] and the prospect of DNA sequencing [21][22][23] based on nucleic acid sensing. [24][25][26][27] The translocation of protein and DNA strands through protein channels is also a biologically relevant process in pathogenic bacteria and human cells.…”

“…Detailed insight has been gained from studies on pores of known molecular structure and the electrophoretically-driven movement of individual DNA and RNA strands. 3,11,21,23,[28][29][30] Several biophysical parameters have been determined such as threading frequency, [31][32][33][34] orientation of strands, [35][36][37][38][39] velocity of DNA transport, 40 influence of pore geometry, 41 and interactions with the pore wall. 39 In addition to nucleic acids, translocation has also been investigated for peptides, [42][43][44][45] proteins, [46][47][48] and peptide-oligonucleotide conjugates, 49 which, unlike nucleic acid strands, frequently feature inhomogeneous charge distributions and vary considerably in diameter along the polymer sequence.…”

First-principles study of high-conductance DNA sequencing with carbon nanotube electrodes

“…1 This discovery initiated a surge of proposals for novel DNA sequencing protocols. 2,3 Among the many, Zwolak et al proposed to sort the nucleobases from each other in a sequence by measuring the transverse tunneling current across a ssDNA as this is driven through a nanopore. 4 It was suggested that single base resolution is, in principle, achievable, as the differences in the electronic structure among the nucleobases may translate into distinguishable tunneling current signals.…”

Placement of Nanospace on an Electrode for Biosensing

“…[14][15][16][17][18][19][20][21][22][23][24] Moreover, the signal, which is based on the native order of the nucleobases in a polynucleotide, can facilitate rapid sequencing of DNA or RNA. [25][26][27][28][29][30][31] One attractive approach to synthetically mimic nature is "molecular imprinting," which is used in the preparation of substrate-selective recognition sites within a matrix using a molecular template. Polyakov 32 reported effects from the presence of organic solvents, such as benzene, toluene, and xylene, on a silica pore structure, and Pauling 33,34 and Dickey 35 later reported on silica gel polymerization using sodium silicate to selectively absorb various organic dyes.…”