A tandem cell for nanopore-based DNA sequencing with exonuclease

Sampath, G.

doi:10.1039/c4ra10326b

Cited by 10 publications

(43 citation statements)

References 28 publications

(93 reference statements)

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“…To remedy this a tandem cell [7] with the structure [cis1, upstream pore (UNP), trans1/cis2, downstream pore (DNP), trans2] and the exonuclease enzyme covalently bonded to the downstream side of UNP was proposed recently. A Fokker-Planck model was used to show that cleaved bases are released in trans1/cis2 without regression into cis1 and, under the right conditions, reach DNP in order.…”

Section: Exonuclease Sequencing Of Dnamentioning

confidence: 99%

“…The Fokker-Planck model developed earlier [7] for the tandem cell can be applied individually to DNP and MNP in the proposed structure. It is a good representation of the diffusion-drift process inside DNP, while inside MNP it is a reasonable approximation.…”

Section: The Present Workmentioning

confidence: 99%

“…Based on the above assumptions the Fokker-Planck model described in [7], which incorporates diffusion and drift (due to electrophoresis and electro-osmotic effects), can be adapted to the structure in Figure 1a. (This model is similar to one based on the original exonuclease sequencing approach [6] and recently extended in [10].)…”

Section: Model Of Cell With Three Stacked Nanopores and Exonucleasementioning

confidence: 99%

“…The Fokker-Planck equation incorporates two kinds of terms, one due to diffusion and the other due to drift [7]. The former corresponds to random movements in 3-d space, while the latter is movement in a directed electric field that is assumed to be constant in the region simulated.…”

Section: Simulation: Assumptions Measures Of Interest Calculationsmentioning

confidence: 99%

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DNA sequencing with stacked nanopores and exonuclease: a simulation-based analysis

Sampath¹

2016

Preprint

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Experiments (Clarke et al., Nat. Nanotech., 2009, 4, 265-270) have shown that DNA could be sequenced using a nanoporebased electrolytic cell in which an exonuclease enzyme in the cis chamber cleaves the leading base of a strand of DNA. The base is identified (with a reported accuracy that exceeds 99%) by the level of the current blockade it causes in the pore; a biological adapter inside slows down the base to lower the detection bandwidth required. This approach, which has been mathematically modeled, analyzed, and simulated (Reiner et al., J. Chem. Phys., 2012, 137, 214903; Brady and Reiner, ibid., 2015 , 143, 074904), is error-prone because bases may be lost to diffusion or enter the pore out of order. Here a modified cell with three stacked nanopores (UNP, MNP, and DNP) and the enzyme attached to the trans side of UNP is proposed. Translocation of a base is simulated with the random walk of a dimensionless particle; the results show that bases translocate through MNP and DNP in sequence order without loss. If this holds in practice then with a suitably designed adapter and compatible enzyme turnover rates base calling accuracy would be limited only by the accuracy of base discrimination. Potential implementation issues are discussed.

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Section: Exonuclease Sequencing Of Dnamentioning

confidence: 99%

Section: The Present Workmentioning

confidence: 99%

Section: Model Of Cell With Three Stacked Nanopores and Exonucleasementioning

confidence: 99%

Section: Simulation: Assumptions Measures Of Interest Calculationsmentioning

confidence: 99%

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DNA sequencing with stacked nanopores and exonuclease: a simulation-based analysis

Sampath¹

2016

Preprint

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“…There is now a growing interest in other kinds of polymers such as polyethylene glycol [2] and proteins/peptides [3,4]. Here a scheme for peptide sequencing is described that is based on a modified version of a tandem cell with cleaving enzymes that was proposed and studied earlier [5,6]. A tandem cell consists of two electrolytic cells joined in tandem and has the structure [cis1, membrane with upstream nanopore (UNP), trans1/cis2, membrane with downstream nanopore (DNP), trans2].…”

Section: Introductionmentioning

confidence: 99%

A quasi-digital approach to peptide sequencing using tandem nanopores with endo- and exo-peptidases

Sampath¹

2015

Preprint

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Abstract.A method of sequencing peptides using tandem cells (RSC Adv., 2015, 5, 167-171; RSC Adv., 2015, 5, 30694-30700) and peptidases is considered. A double tandem cell (two tandem cells in tandem) has three nanopores in series, an amino-acid-specific endopeptidase attached downstream of the first pore, and an exopeptidase attached downstream of the second pore. The endopeptidase cleaves a peptide threaded through the first pore into fragments that are well separated in time. Fragments pass through the second pore and are each cleaved by the exopeptidase into a series of single residues; the latter pass through the third pore and cause separate current blockades that can be counted. This leads to an ordered list of integers corresponding to the number of residues in each fragment. With 20 cells, one per amino acid type, and 20 peptide copies, the resulting 20 lists of integers are used by a simple algorithm to assemble the sequence. This is a quasi-digital process that uses the lengths of subsequences to sequence the peptide, it differs from conventional analog methods which seek to identify monomers in a polymer via differences in blockade levels, residence times, or transverse currents. Several implementation issues are discussed. In particular the problem of fast analyte translocation, widely considered intransigent, may be resolved through the use of a sufficiently long (40-60 nm) third pore. This translates to a required bandwidth of 1-2 MHz, which is within the range of currently available CMOS circuits. IntroductionNanopores have been investigated over several years for their potential use in the analysis of polymers, in particular DNA [1]. There is now a growing interest in other kinds of polymers such as polyethylene glycol [2] and proteins/peptides [3,4]. Here a scheme for peptide sequencing is described that is based on a modified version of a tandem cell with cleaving enzymes that was proposed and studied earlier [5,6]. A tandem cell consists of two electrolytic cells joined in tandem and has the structure [cis1, membrane with upstream nanopore (UNP), trans1/cis2, membrane with downstream nanopore (DNP), trans2]. An enzyme attached to the downstream side of UNP successively cleaves the leading monomer in a polymer that has threaded through UNP; the cleaved monomer then translocates to and through DNP where the resulting ionic current blockade level (and/or other discriminators [6]) is used to identify it. With DNA the enzyme is an exonuclease [5], with peptides it is an amino or carboxy peptidase [6]. The approach does not use labels or immobilization.The present communication describes a method of peptide sequencing using a double tandem cell (two tandem cells connected in tandem), an endopeptidase to break the peptide into fragments, and an exopeptidase to obtain the lengths of the latter. With 20 such cells these length data are used by a simple algorithm to assemble the sequence.

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DNA sequencing with stacked nanopores and exonuclease: A simulation‐based analysis

Sampath

2016

Electrophoresis

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Experiments have shown that DNA can be sequenced using an electrolytic cell with a nanopore and an exonuclease enzyme in the cis chamber that cleaves the leading mononucleotide in a strand of DNA. The base therein can be identified with an accuracy of 80-90% by the level of the current blockade caused in the pore; a biological adapter inside slows down the cleaved mononucleotide and lowers the detection bandwidth required. In this approach, which has been mathematically modeled, analyzed, and simulated, mononucleotides are likely to be lost to diffusion or enter the pore out of order. To remedy this, a modified cell with three stacked nanopores (named UNP, MNP, and DNP) and the enzyme attached to the trans side of UNP is proposed and modeled. Mononucleotide translocation is simulated with the random walk of a dimensionless particle; the results show that the cleaved mononucleotides translocate through MNP and DNP in sequence order without loss. If this holds in practice then with a suitably designed adapter and compatible enzyme turnover rates sequencing accuracy would be limited only by the accuracy of mononucleotide discrimination. Potential implementation issues are discussed.

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A tandem cell for nanopore-based DNA sequencing with exonuclease

Cited by 10 publications

References 28 publications

DNA sequencing with stacked nanopores and exonuclease: a simulation-based analysis

DNA sequencing with stacked nanopores and exonuclease: a simulation-based analysis

A quasi-digital approach to peptide sequencing using tandem nanopores with endo- and exo-peptidases

DNA sequencing with stacked nanopores and exonuclease: A simulation‐based analysis

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