scholarly journals Design and characterization of a nanopore-coupled polymerase for single-molecule DNA sequencing by synthesis on an electrode array

2016 ◽  
Vol 113 (44) ◽  
pp. E6749-E6756 ◽  
Author(s):  
P. Benjamin Stranges ◽  
Mirkó Palla ◽  
Sergey Kalachikov ◽  
Jeff Nivala ◽  
Michael Dorwart ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Dna Synthesis ◽  
Lipid Bilayers ◽  
High Throughput ◽  
Single Molecule ◽  
Low Cost ◽  
Electrode Array ◽  
Oxide Semiconductor ◽  
Sequencing Platform ◽  
Sequencing By Synthesis

Scalable, high-throughput DNA sequencing is a prerequisite for precision medicine and biomedical research. Recently, we presented a nanopore-based sequencing-by-synthesis (Nanopore-SBS) approach, which used a set of nucleotides with polymer tags that allow discrimination of the nucleotides in a biological nanopore. Here, we designed and covalently coupled a DNA polymerase to an α-hemolysin (αHL) heptamer using the SpyCatcher/SpyTag conjugation approach. These porin–polymerase conjugates were inserted into lipid bilayers on a complementary metal oxide semiconductor (CMOS)-based electrode array for high-throughput electrical recording of DNA synthesis. The designed nanopore construct successfully detected the capture of tagged nucleotides complementary to a DNA base on a provided template. We measured over 200 tagged-nucleotide signals for each of the four bases and developed a classification method to uniquely distinguish them from each other and background signals. The probability of falsely identifying a background event as a true capture event was less than 1.2%. In the presence of all four tagged nucleotides, we observed sequential additions in real time during polymerase-catalyzed DNA synthesis. Single-polymerase coupling to a nanopore, in combination with the Nanopore-SBS approach, can provide the foundation for a low-cost, single-molecule, electronic DNA-sequencing platform.

scholarly journals Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array

2016 ◽  
Vol 113 (19) ◽  
pp. 5233-5238 ◽  
Author(s):  
Carl W. Fuller ◽  
Shiv Kumar ◽  
Mintu Porel ◽  
Minchen Chien ◽  
Arek Bibillo ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Real Time ◽  
Lipid Bilayers ◽  
Dna Polymerase ◽  
Single Molecule ◽  
High Throughput Sequencing ◽  
Electrical Current ◽  
Sequencing Data ◽  
Sequencing By Synthesis ◽  
Dna Strand

DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5′-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods.

Controllable and reversible DNA translocation through a single-layer molybdenum disulfide nanopore

Nanoscale ◽  
2018 ◽  
Vol 10 (41) ◽  
pp. 19450-19458 ◽  
Author(s):  
Wei Si ◽  
Yin Zhang ◽  
Jingjie Sha ◽  
Yunfei Chen
Keyword(s):  
Dna Sequencing ◽  
Molybdenum Disulfide ◽  
High Throughput ◽  
Low Cost ◽  
Single Layer ◽  
Dna Translocation ◽  
Nanopore Dna Sequencing

A challenge that remains to be solved in the high-throughput and low-cost nanopore DNA sequencing is that DNA translocates through the nanopore too quickly to be sequenced with enough accuracy.

scholarly journals Hi-Plex2: a simple and robust approach to targeted sequencing-based genetic screening

BioTechniques ◽  
2019 ◽  
Vol 67 (3) ◽  
pp. 118-122 ◽  
Author(s):  
Fleur Hammet ◽  
Khalid Mahmood ◽  
Thomas R Green ◽  
Tu Nguyen-Dumont ◽  
Melissa C Southey ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Dna Sequencing ◽  
Multiplex Pcr ◽  
High Throughput ◽  
Genetic Screening ◽  
Low Cost ◽  
Substantial Reduction ◽  
Targeted Sequencing ◽  
Robust Approach ◽  
Panel Size

We have previously reported Hi-Plex, a multiplex PCR methodology for building targeted DNA sequencing libraries that offers a low-cost protocol compatible with high-throughput processing. Here, we detail an improved protocol, Hi-Plex2, that more effectively enables the robust construction of small-to-medium panel-size libraries while maintaining low cost, simplicity and accuracy benefits of the Hi-Plex platform. Hi-Plex2 was applied to three panels, comprising 291, 740 and 1193 amplicons, targeting genes associated with risk for breast and/or colon cancer. We show substantial reduction of off-target amplification to enable library construction for small-to-medium-sized design panels not possible using the previous Hi-Plex chemistry.

scholarly journals An ISFET Microarray Sensor System for Detecting the DNA Base Pairing

Micromachines ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 731
Author(s):  
Peng Sun ◽  
Yongxin Cong ◽  
Ming Xu ◽  
Huaqing Si ◽  
Dan Zhao ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Large Scale ◽  
Ph Value ◽  
Low Cost ◽  
Complementary Metal Oxide Semiconductor ◽  
Fluorescent Labeling ◽  
Oxide Semiconductor ◽  
Sensor System ◽  
Base Pairing ◽  
Dna Base

Deoxyribonucleic acid (DNA) sequencing technology provides important data for the disclosure of genetic information and plays an important role in gene diagnosis and gene therapy. Conventional sequencing devices are expensive and require large and bulky optical structures and additional fluorescent labeling steps. Sequencing equipment based on a semiconductor chip has the advantages of fast sequencing speed, low cost and small size. The detection of DNA base pairing is the most important step in gene sequencing. In this study, a large-scale ion-sensitive field-effect transistor (ISFET) array chip with more than 13 million sensitive units is successfully designed for detecting the DNA base pairing. DNA base pairing is successfully detected by the sensor system, which includes the ISFET microarray chip, microfluidic system, and test platform. The chip achieves a high resolution of at least 0.5 mV, thus enabling the recognition of the change of 0.01 pH value. This complementary metal-oxide semiconductor (CMOS) compatible and cost-efficient sensor array chip, together with other specially designed components, can form a complete DNA sequencing system with potential application in the molecular biology fields.

scholarly journals Assessing protein dynamics on low complexity single-strand DNA curtains

2018 ◽  
Author(s):  
Jeffrey M. Schaub ◽  
Hongshan Zhang ◽  
Michael M. Soniat ◽  
Ilya J. Finkelstein
Keyword(s):  
Lipid Bilayers ◽  
High Throughput ◽  
Single Molecule ◽  
Binding Proteins ◽  
Low Complexity ◽  
Rolling Circle Replication ◽  
Sequence Composition ◽  
Ssdna Binding ◽  
Length Changes ◽  
Ssdna Binding Proteins

AbstractSingle-stranded DNA (ssDNA) is a critical intermediate in all DNA transactions. As ssDNA is more flexible than double-stranded (ds)DNA, interactions with ssDNA-binding proteins (SSBs) may significantly compact or elongate the ssDNA molecule. Here, we develop and characterize low-complexity ssDNA curtains, a high-throughput single-molecule assay to simultaneously monitor protein binding and correlated ssDNA length changes on supported lipid bilayers. Low-complexity ssDNA is generated via rolling circle replication of short synthetic oligonucleotides, permitting control over the sequence composition and secondary structure-forming propensity. One end of the ssDNA is functionalized with a biotin, while the second is fluorescently labeled to track the overall DNA length. Arrays of ssDNA molecules are organized at microfabricated barriers for high-throughput single-molecule imaging. Using this assay, we demonstrate that E. coli SSB drastically and reversibly compacts ssDNA templates upon changes in NaCl concentration. We also examine the interactions between a phosphomimetic RPA and ssDNA. Our results indicate that RPA-ssDNA interactions are not significantly altered by these modifications. We anticipate low-complexity ssDNA curtains will be broadly useful for single-molecule studies of ssDNA-binding proteins involved in DNA replication, transcription and repair.

scholarly journals VLSI Structures for DNA Sequencing—A Survey

2020 ◽  
Vol 7 (2) ◽  
pp. 49
Author(s):  
Mohammad A. Islam ◽  
Palash K. Datta ◽  
Harley Myler
Keyword(s):  
Dna Sequencing ◽  
High Throughput ◽  
Biomedical Research ◽  
Field Effect ◽  
Field Effect Transistors ◽  
Implementation Strategies ◽  
Metal Oxide Semiconductor ◽  
Oxide Semiconductor ◽  
New Approach ◽  
Technical Advances

DNA sequencing is a critical functionality in biomedical research, and technical advances that improve it have important implications for human health. Novel methods by which sequencing can be accomplished in more accurate, high-throughput, and faster ways are in development. Here, we review VLSI biosensors for nucleotide detection and DNA sequencing. Implementation strategies are discussed and split into function-specific architectures that are presented for reported design examples from the literature. Lastly, we briefly introduce a new approach to sequencing using Gate All-Around (GAA) nanowire Metal Oxide Semiconductor Field Effect Transistors (MOSFETs) that has significant implications for the field.

scholarly journals Low-cost, bottom-up fabrication of large-scale single-molecule nanoarrays by DNA origami placement

2020 ◽  
Author(s):  
Rishabh M. Shetty ◽  
Sarah R. Brady ◽  
Paul W. K. Rothemund ◽  
Rizal F. Hariadi ◽  
Ashwin Gopinath
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Large Scale ◽  
Low Cost ◽  
Maximum Yield ◽  
Dna Origami ◽  
Top Down ◽  
Statistical Limit ◽  
Macro Scale ◽  
Self Assembled

Large-scale nanoarrays of single biomolecules enable high-throughput assays while unmasking the underlying heterogeneity within ensemble populations. Until recently, creating such grids which combine the unique advantages of microarrays and single-molecule experiments (SMEs) has been particularly challenging due to the mismatch between the size of these molecules and the resolution of top-down fabrication techniques. DNA Origami Placement (DOP) combines two powerful techniques to address this issue: (i) DNA origami, which provides a ∼ 100-nm self-assembled template for single-molecule organization with 5 nm resolution, and (ii) top-down lithography, which patterns these DNA nanostructures, transforming them into functional nanodevices via large-scale integration with arbitrary substrates. Presently, this technique relies on state-of-the-art infrastructure and highly-trained personnel, making it prohibitively expensive for researchers. Here, we introduce a bench-top technique to create meso-to-macro-scale DNA origami nanoarrays using self-assembled colloidal nanoparticles, thereby circumventing the need for top-down fabrication. We report a maximum yield of 74%, two-fold higher than the statistical limit of 37% imposed on non-specific molecular loading alternatives. Furthermore, we provide a proof-of-principle for the ability of this nanoarray platform to transform traditionally low-throughput, stochastic, single-molecule assays into high-throughput, deterministic ones, without compromising data quality. Our approach has the potential to democratize single-molecule nanoarrays and demonstrates their utility as a tool for biophysical assays and diagnostics.

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