Nanopore DNA sequencing with MspA

Abstract

Nanopore sequencing has the potential to become a direct, fast, and inexpensive DNA sequencing technology. The simplest form of nanopore DNA sequencing utilizes the hypothesis that individual nucleotides of single-stranded DNA passing through a nanopore will uniquely modulate an ionic current flowing through the pore, allowing the record of the current to yield the DNA sequence. We demonstrate that the ionic current through the engineered Mycobacterium smegmatis porin A, MspA, has the ability to distinguish all four DNA nucleotides and resolve single-nucleotides in single-stranded DNA when double-stranded DNA temporarily holds the nucleotides in the pore constriction. Passing DNA with a series of double-stranded sections through MspA provides proof of principle of a simple DNA sequencing method using a nanopore. These findings highlight the importance of MspA in the future of nanopore sequencing.bionanotechnology | next generation sequencing | single-molecule | stochastic sensing | protein pore T he information encoded in DNA is of paramount importance to medicine and to the life sciences. The mapping of the human genome is revolutionizing the understanding of genetic disorders and the prediction of disease and will aid in developing therapies as in refs. 1-3. The ability to sequence DNA quickly and inexpensively is essential to individualized medicine and to scientific research and has prompted the development of new sequencing techniques beyond the original Sanger sequencing (4-7). Nanopore DNA sequencing represents one of the approaches being developed to rapidly sequence a human genome for under $1,000 (www.genome.gov/12513210).In the most elementary form of nanopore DNA sequencing, a nanometer-scale pore provides the sole pathway for an ionic current. Single-stranded DNA (ssDNA) is electrophoretically driven through the pore, and as the ssDNA passes through, it reduces the ionic current through the pore. If each passing nucleotide yields a characteristic residual ionic current then the record of the current will correspond to the DNA sequence. This simple and reagent-free sequencing technique holds the promise to inexpensively read long lengths of DNA molecules at intrinsically fast rates (8). Due to its inherently small size, this system is amenable to parallelization (9).Lately, nanopore sequencing techniques have progressed substantially. This progress and the remaining challenges in nanopore DNA sequencing are summarized in a review article by Branton et al. (8). While nanotechnology usually involves materials such as Si and SiN, nanopore DNA sequencing first evolved using the well-studied protein porin α-hemolysin (10). In contrast to pores made from inorganic materials (11, 12), protein pores can be easily modified and produced with repeatable subnanometer accuracy. Stoddart (13) and Purnell (14) demonstrated that several locations within the beta barrel of α-hemolysin exhibit nucleotide-specific sensitivity with immobilized ssDNA (13). However, α-hemolysin's 5 nm-long cylindrical beta barrel presents...

Controlling polymer capture and translocation by electrostatic polymer-pore interactions

“…The physical mechanism behind this peculiarity is encoded in the force balance Eq. (9). One sees that the electric field E acts on the whole polymer with length L p whereas the barrier-induced force −V p (z p ) is induced exclusively by the polymer portion l p located in the pore.…”

“…(8) we eliminate the term u c (a) in Eq. (9). Accounting also for Gauss' law φ (a) = 4π B σ p , after some algebra the polymer velocity follows as…”

“…In this context, nanopore-based sequencing approaches have been a central focus over the past two decades. Polymer translocation was initially conceptualised by using biological nanopores such as α-Hemolysin channels of limited characteristics and undesirable fragility [1][2][3][4][5][6][7][8][9]. Recent advancements in nanotechnology have significantly improved the reliability of the sequencing techniques.…”

Interfacing solid‐state nanopores with gel media to slow DNA translocations

“…In fact, combining enzymes with an MspA nanopore [45] enabled single-nucleotide resolution [46] of ssDNA strands.…”

Fabrication and Healing of Faceted Nanopores in Magnesium

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Solid-state nanopores have been extensively investigated due to many biological engineering and scientific applications, such as for rapid electrical detection and analysis of biopolymers, powerful DNA detection and genome sequencing technology [1][2]. Herein, we demonstrate that faceted nanopores with various shapes can be successfully fabricated in magnesium via focused electron beam (e-beam) technology inside transmission electron microscope (TEM).

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