Mapping and sequencing DNA using nanopores and nanodetectors

Abstract

Even prior to the introduction of capillary DNA sequencers, nanopores were discussed as a low-cost, high-throughput substrate for sequencing. Since then, other next-generation sequencing technologies have been developed and achieved widespread use, but nanopores have lagged behind due to difficulties in generating usable sequence data. The practical and theoretical issues of translocation speed and signal detection encountered when attempting to sequence DNA with nanopores are discussed. Various methods that different laboratories have used to overcome difficulties in biologically based and solid-state nanopores are also presented. Different approaches designed to circumvent the overriding issue of detecting signals from individual bases in a time-resolved manner in nanopores are described. For example, genomic positional sequencing utilizes hybridization of short oligonucleotide probes to very long DNA templates and then detects these probes by variations in current blockade in solid-state nanodetectors. The positions of the probes relative to each other and relative to the ends of the DNA are determined by measuring the time between current blockade peaks. By assembling many such measurements, it is possible to overcome the problems encountered when attempting to sequence DNA at high speed in nanopores, providing the potential for true de novo sequencing of large genomes on a routine basis.

Smooth DNA Transport through a Narrowed Pore Geometry

“…Despite ample promise, two major shortcomings of nanopores with respect to dsDNA analysis have been 1) Mean transport speeds are 10-100 times faster than required for statistical averaging of ion current data from short DNA regions inside the pore (5), and 2) DNA position versus time in the pore is not well known or otherwise regulated (3,25,26). Although regulation has been achieved for single-stranded DNA using enzymes as molecular stepper motors (9,10,(27)(28)(29)(30)(31), regulated motion of dsDNA has yet to be demonstrated.…”

“…Voltage-driven dsDNA translocation through 5-15 nm diameter pores in thin (20-50 nm) solid-state materials proceeds with mean velocities of 10-100 ns/bp, and often in large pores multiple DNA strands enter simultaneously (17,19,32,33), which complicates single-file readout of information that is encoded in the linear sequence. Proteins such as RecA from Escherichia coli form filaments around the DNA that slows DNA transport and prevents its folding (26,34,35), although this approach inherently masks chemical information contained within the DNA, such as the presence of DNA chemical modifications (7,36) or small bound drug/reporter molecules (37)(38)(39)(40). Explorations of the effects of parameters such as the electrolyte viscosity (41,42), salt type (43,44), membrane material (45,46), applied pressure (47,48), and chemical composition inside (49)(50)(51)(52) and outside (53) the pore have yielded only moderate DNA retardation factors.…”

Single‐molecule bioelectronics

“…However, while signal-to-noise ratios for counting single molecules can be quite robust, resolving features within individual molecules is often somewhat less reliable. 33 …”

“…(Additionally, there are ongoing commercial efforts which incorporate ASICs with nanopore measurements 2,33 , although at this time few specific technical details are publically available.) Integrated amplifier designs face similar tradeoffs as discrete designs, with different constraints.…”

Overview of High Throughput Sequencing Technologies to Elucidate Molecular Pathways in Cardiovascular Diseases

“…Nanopore sequencing technologies comprise a relatively newer set of techniques being developed by companies such as Oxford Nanopore and NABsys (http://www.nabsys.com/), which are working on massively parallel sequencers not based on sequencing by synthesis. The concept is to electrophorese molecules through a pore of a membrane and then measure the electrical current through the pore as molecules pass through 93 (Figure 5). By characterizing the current of a pore over time, nanopore technology may be able to determine exactly what has traversed the pore and in which order.…”