Single-Molecule Electrical Random Resequencing of DNA and RNA

Abstract

Two paradigm shifts in DNA sequencing technologies—from bulk to single molecules and from optical to electrical detection—are expected to realize label-free, low-cost DNA sequencing that does not require PCR amplification. It will lead to development of high-throughput third-generation sequencing technologies for personalized medicine. Although nanopore devices have been proposed as third-generation DNA-sequencing devices, a significant milestone in these technologies has been attained by demonstrating a novel technique for resequencing DNA using electrical signals. Here we report single-molecule electrical resequencing of DNA and RNA using a hybrid method of identifying single-base molecules via tunneling currents and random sequencing. Our method reads sequences of nine types of DNA oligomers. The complete sequence of 5′-UGAGGUA-3′ from the let-7 microRNA family was also identified by creating a composite of overlapping fragment sequences, which was randomly determined using tunneling current conducted by single-base molecules as they passed between a pair of nanoelectrodes.

Statistical Inference for Nanopore Sequencing with a Biased Random Walk Model

“…An experiment similar to this model was demonstrated on very short sequences with tunneling current data in Ohshiro et al (11). Here, we extend the approach to the longer sequences expected from a nanopore device.…”

The emergence of nanopores in next-generation sequencing

“…They aim to combine biological nanopores with an optical detection method. Quantum Biosystems is commercializing research by Prof. T Kawai, who combined a tunneling electron detector with a nanopore to sequence DNA [37]. Base4, on the other hand, aims to cleave single nucleotides into droplets in a water-oil emulsion and detect their presence by a chemical cascade reaction.…”

Single-Molecule Analysis Methods Using Nanogap Electrodes and Their Application to DNA Sequencing Technologies

“…Single-molecule analysis methods, and in particular single-molecule conductance measurements, have been used to tell individual base molecules in DNA and RNA apart from one another in aqueous solutions, and they have allowed for base sequences of DNA and RNA to be determined. 3,36,37 Single-molecule analysis methods have also been used to identify modified base molecules known as disease markers, amino acids, and partial sequences of amino acids in peptides. 3,38 Because the analysis of biopolymers at single-molecule resolutions is expected to lead to the development of highprecision and high-throughput DNA sequencing technologies, 3,93 several single-molecule measurement methods have been proposed; however, in this chapter we will focus only on the method that uses nano-MCBJs, objective scientific analysis.…”

“…Ideally, the base molecules would be analyzed one-by-one as they pass between nanogap electrodes (Figure 20). 37 In order to develop this analytical method, it is necessary to develop the following two methods first: (1) a method for identifying single-base molecular species using single-molecule conductance; 3 and (2) a method for controlling the structure and flow rate of a strand of DNA. 96,97 In this sub-chapter, we focus only on how to identify individual base molecules using conductance.…”

“…3 The earliest methods focused on base molecules with π electron systems, as these would allow a large amount of electricity to flow through the single molecules and allow for the base molecules to be identified at single-molecule resolutions; this was considered to be an important step in the sequencing of individual strands of DNA and RNA. 31 36 Currently, it is possible to determine sequences of base molecules in DNA and RNA 37 and partial sequences of amino acids in peptides. 38,39 Moreover, because single-molecule conductance strongly corresponds with the properties of single molecules, single-molecule analysis methods can identify the chemically modified base molecules that traditionally denote cancer or diseases markers; this is despite current DNA sequencing technologies not being able to identify the base molecules directly.…”