Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array
| Autor: | James Pollard, Zengmin Li, Ashwini Bhat, Andrew Trans, Jennifer Hovis, Minchen Chien, Cleoma Arnold, Edward Shian Liu, Arek Bibillo, Randy Davis, John J. Kasianowicz, Mirko Palla, James J. Russo, Mintu Porel, Irina Morozova, Wenjing Guo, Jingyue Ju, Shundi Shi, Cynthia Cech, Shiv Kumar, Eric Takeshi Harada, Chuanjuan Tao, Carl W. Fuller, Alexander Yang, Michael Dorwart, Stefan Roever, Roger Chen, Anne Aguirre, George M. Church, Daniel Korenblum, P. Benjamin Stranges, Sergey Kalachikov |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Conductometry Polymers DNA polymerase Nanotechnology 010402 general chemistry 01 natural sciences DNA sequencing Nanopores 03 medical and health sciences chemistry.chemical_compound Computer Systems Precision Medicine Polymerase Oligonucleotide Array Sequence Analysis Multidisciplinary Base Sequence Staining and Labeling biology Nucleotides Oligonucleotide DNA Equipment Design Sequence Analysis DNA Biological Sciences 0104 chemical sciences Equipment Failure Analysis Nanopore 030104 developmental biology chemistry biology.protein Biophysics Nucleic acid Primer (molecular biology) |
| Zdroj: | Proceedings of the National Academy of Sciences. 113:5233-5238 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.1601782113 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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