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Real-Time, Single Molecule Electronic DNA Sequencing by Synthesis Using Polymer Tagged Nucleotides on a Nanopore Array

Published

Author(s)

Carl Fuller, Shiv Kumar, Mintu Porel, Michen Chien, Arek Bibillo, P. B. Stranges, Michael Dorwart, Chuanjua Tao, Zengmin Li, Wenjing Guo, Shundi Shi, Daniel Korenblum, Andrew Trans, Anne Aguirre, Edward Liu, Takeshi Harada, James Pollard, Ashwini Bhat, Cynthia Cech, Alexander Yang, Cleoma Spilman, Mirko Palla, Jennifer Hovis, Roger Chen, Irina Morozova, Sergey Kalachikov, James Russo, John J. Kasianowicz, Stefan Roever, George Church, Jingyue Ju

Abstract

DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we developed a single molecule nanopore-based SBS strategy (NanoSBS) that accurately distinguishes four bases by electronically detecting and differentiating 4 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 NanoSBS on an α-hemolysin nanopore array platform. We synthesized several polymer-tagged nucleotides using tags that produce different blockade current levels, and verified they are active substrates for DNA polymerase. Highly processive DNA polymerases were conjugated to the nanopores, and single nanopore-polymerase conjugates were complexed to primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When a nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and current level is measured. The levels displayed with differently tagged nucleotides captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, and real-time single molecule electronic DNA sequencing data with single base resolution was obtained. The use of these novel tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, could lead to new high throughput sequencing methods.
Citation
Proceedings of the National Academy of Sciences of the United States of America

Keywords

Nanopore, Single Molecule Sequencing, DNA Sequencing by Synthesis

Citation

Fuller, C. , Kumar, S. , Porel, M. , Chien, M. , Bibillo, A. , Stranges, P. , Dorwart, M. , Tao, C. , Li, Z. , Guo, W. , Shi, S. , Korenblum, D. , Trans, A. , Aguirre, A. , Liu, E. , Harada, T. , Pollard, J. , Bhat, A. , Cech, C. , Yang, A. , Spilman, C. , Palla, M. , Hovis, J. , Chen, R. , Morozova, I. , Kalachikov, S. , Russo, J. , Kasianowicz, J. , Roever, S. , Church, G. and Ju, J. (2016), Real-Time, Single Molecule Electronic DNA Sequencing by Synthesis Using Polymer Tagged Nucleotides on a Nanopore Array, Proceedings of the National Academy of Sciences of the United States of America (Accessed December 14, 2024)

Issues

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Created May 9, 2016, Updated October 12, 2021