PEG-Labeled Nucleotides and Nanopore Detection for Single Molecule DNASequencing by Synthesis

Kumar, Shiv; Tao, Chuanjuan; Chien, Minchen; Hellner, Brittney; Balijepalli, Arvind; Robertson, Joseph W. F.; Li, Zengmin; Russo, James J.; Reiner, Joseph E.; Kasianowicz, John J.; Ju, Jingyue

doi:10.1038/srep00684

Cited by 112 publications

(106 citation statements)

References 41 publications

(64 reference statements)

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“…这种基于纳米孔道的 DNA 单分子测序分析方法因其快速、无需 PCR 扩增、无需荧光标记就可直接获取碱基序列信息而被视为最具前景的"第三代 DNA 测序技术"(Third-Generation DNA Sequencing), 有望实现仅需 1000 美金、24 h 内测定个人基因组的完整序列 [5] . [6] ; 用不同长度的聚乙二醇分子标记不同种类的碱基, 从而增强不同碱基的电流信号差异 [7] ; . 例如, 分枝杆菌属孔道蛋白 A(MspA)由于其孔道最窄处的厚度小(长约 0.5 nm), 孔径窄(直径约 1.2 nm), 相比于 α-溶血素对每个碱基(A, T, C 和 G)的分辨表现出更高的灵敏度 [10] .…”

Section: 引言unclassified

Detection of Single Oligonucleotide by an Aerolysin Nanopore

Cao¹,

Liao²,

Ying³

et al. 2016

Acta Chim. Sinica

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Section: 引言unclassified

Detection of Single Oligonucleotide by an Aerolysin Nanopore

Cao¹,

Liao²,

Ying³

et al. 2016

Acta Chim. Sinica

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“…In addition, both of these methods rely on distinctions among the four natural bases, two relatively similar purines and two similar pyrimidines. Our nanopore SBS approach has the advantage of using synthetic polymer tags attached to the nucleotides that are designed specifically to produce unique and readily distinguishable ionic current blockade signatures for sequence determination (11). Direct attachment of the polymerase to the nanopore ensures that there is ample time for the polymer tags on the incoming nucleotide to be captured in the nanopore before completion of the DNA polymerase catalytic cycle.…”

Section: Significancementioning

confidence: 99%

“…sequencing in which nucleotides are enzymatically cleaved one-byone from a DNA molecule and monitored as they are captured by and pass through the nanopore (10), and a nanopore SBS approach, described by us, in which identifiable polymer tags are attached to nucleotides and registered in nanopores during enzyme-catalyzed DNA synthesis (11). Common to all these methods is the need for precise control of the reaction rates so that each base is determined in order and without fail.…”

Section: Significancementioning

confidence: 99%

See 1 more Smart Citation

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

Fuller

Kumar

Porel

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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122

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DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a singlemolecule 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 oligonucleotidebased 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 singlemolecule 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.single-molecule sequencing | nanopore | DNA sequencing by synthesis | polymer-tagged nucleotides | chip array T he importance of DNA sequencing has increased dramatically from its inception four decades ago. It is recognized as a crucial technology for most areas of biology and medicine and as the underpinning for the new paradigm of personalized and precision medicine. Information on individuals' genomes and epigenomes can help reveal their propensity for disease, clinical prognosis, and response to therapeutics, but routine application of genome sequencing in medicine will require comprehensive data delivered in a timely and cost-effective manner (1). Although 35 years of technological advances have improved sequence throughput and have reduced costs exponentially, genome analysis still takes several days and thousands of dollars to complete (1, 2). To realize the potential of personalized medicine fully, the speed and cost of sequencing must be brought down another order of magnitude while increasing sequencing accuracy and read length. Singlemolecule approaches are thought to be essential to meet these requirements and offer the additional benefit of eliminating amplification bias (3, 4). Although optical methods for singlemolecule sequencing have been achieved and commercialize...

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“…Recently, we reported a method for SBS with nanopore detection (19,20). This approach has two distinct features: the use of nucleotides with specific tags to enhance base discrimination and a ternary DNA polymerase complex to hold the tagged nucleotides long enough for tag recognition by the nanopore.…”

mentioning

confidence: 99%

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

Stranges

Palla

Kalachikov

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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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.nanopore sequencing | protein design | polymer-tagged nucleotides | single-molecule detection | integrated electrode array D NA sequencing is a fundamental technology in the biological and medical sciences (1). Advances in sequencing technology have enabled the growth of interest in individualized medicine with the hope of better treating human disease. The cost of genome sequencing has dropped by five orders of magnitude over the last decade but still remains out of reach as a conventional clinical tool (2, 3). Thus, the development of new, high-throughput, accurate, low-cost DNA-sequencing technologies is a high priority. Ensemble sequencing-by-synthesis (SBS) platforms dominate the current landscape. During SBS, a DNA polymerase binds and incorporates a nucleotide analog complementary to the template strand. Depending on the instrumentation, this nucleotide is identified either by its associated label or the appearance of a chemical by-product upon incorporation (4). These platforms take advantage of a high-fidelity polymerase reaction but require amplification and have limited read lengths (5). Recently, single-molecule strategies have been shown to have great potential to achieve long read lengths, which is critical for highly scalable and reliable genomic analysis (6-9). Pacific Biosciences' SMRT SBS approach has been used for this purpose but has lower throughput and higher cost compared with current secondgeneration technology (10).Since the first demonstration of single-molecule characterization by a biological nanopore two decades ago (11), interest has grown in using nanopores as sensors for DNA base discrimination. One appro...

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PEG-Labeled Nucleotides and Nanopore Detection for Single Molecule DNASequencing by Synthesis

Cited by 112 publications

References 41 publications

Detection of Single Oligonucleotide by an Aerolysin Nanopore

Detection of Single Oligonucleotide by an Aerolysin Nanopore

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

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

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