Nanopore-based fourth-generation DNA sequencing technology

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.

Keywords: DNA sequencing; Nanopore; Single base; Single molecule.

Figure 1

Schematic view of nanopore fluidic setup comprising a DNA molecule through the nanopore PMMA stands for polymethyl methacrylate.

Figure 2

Side and top views of three biological nanopores A. Heptameric α-hemolysin toxin from Staphylococcus aureus, figure adapted from . B. Octameric MspA porin from Mycobacterium smegmatis, figure adapted from . C. Dodecamer connector channel from bacteriophage phi29 DNA packaging motor, figure adapted from .

Figure 3

The portable MinION –– the first handheld nanopore DNA sequencer This figure was reproduced from with permission.

Figure 4

Solid state nanopores A. Top view of single 2-nm diameter nanopore in silicon nitride membrane. B. Arrays of 18-nm nanopores in diameters with single conical angle 12.7 ± 5.4° (N = 19) fabricated with standard semiconductor process. C. 200-mm wafers patterned in a matrix of 11 × 11 3-metal nanopore devices. The dark field TEM images are presented in D and E and the EELS element analysis results along the red solid lines in D and E are presented in F and G, respectively. Panels B–G were reproduced from with permission. TEM, transmission electron microscopy; EELS, electron energy loss spectroscopy.

Figure 5

Experimental results on ssDNA translocation through a 6-nm nanopore A. Current trace for ssDNA in water. B. Current trace for ssDNA in a 50% glycerol solution. C. Scatter plots (blockade current vs. dwell time) for current signals of ssDNA translocation in water (black triangles), 20% glycerol (red dots) and 50% glycerol (blue squares) solutions. Insets in A and B show typical translocation events. This figure was reproduced from with permission. ssDNA, single-stranded DNA.

Figure 6

Experimental set-up A. A schematic of DNA translocation through a SAM-coated nanopore. B. and C. TEM images of a 12-nm diameter solid-state nanopore before (B) and after (C) coating with carboxyl benzyl phosphonic acid, respectively. Red circles highlight pore sizes. D. Configurations of molecules in the hydrophilic and hydrophobic SAMs. E. Translocation of DNA through the same nanopore coated with hydrophilic SAMs at 300 mV. F. Hydrophobic-SAM coating, switched from the hydrophilic-SAM coating after adding trimethylsilyl diazomethane. G. and H. Typical ionic current signals of DNA translocation events at hydrophilic and hydrophobic states, respectively. Figure was reproduced from with permission. SAM, self-assembled monolayer.

Figure 7

Effects of peptide length and structure on the transport of peptides through a single (M113Y)₇ pore A. Representative single-channel traces. B. Current blockage amplitudes. C. Event mean dwell times. The experiments were performed under a series of symmetrical buffer conditions with a 2.0 ml solution comprising 1 M NaCl and 10 mM Tris–HCl (pH 7.5) at +50 mV (cis at ground). Peptides were added to the trans compartment, while the (M113Y)₇ protein was added from cis of the chamber device. The final concentrations of peptides in the buffer were 1.0 μM each. Figure was reproduced from with permission.

Figure 8

Molecular graphic representation of the staphylococcal α-HL protein with β-CD lodged in the lumen of the channel A. Side view of the (M113F/K147N)₇ pore. B. Top view into the (M113F/K147N)₇ pore from the cis side of the lipid bilayer, highlighting positions 113 (orange) and 147 (cyan), where the naturally-occurring Met and Lys residues have been substituted with Phe and Asn, respectively. β-CD molecule is shown in red. C. Structures of PMPA and CMPA. The detection of PMPA and CMPA was shown by the typical single-channel current recording traces. D. PMPA/CMPA was not detected. E. 2 μM PMPA was detected. F. 2 μM CMPA was detected. The experiments were performed at −80 mV in 1 M NaCl and 10 mM Tris–HCl (pH 7.5) in the presence of 40 μM β-CD. Figure was reproduced from with permission. α-HL, α-hemolysin; β-CD, β-cyclodextrin; PMPA, pinacolyl methylphosphonic acid; CMPA, cyclohexyl methylphosphonic acid.