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

作者：Carl W. Fuller, Shiv Kumar, Mintu Porel, Minchen Chien, Arek Bibillo, P. Benjamin Stranges, Michael R. Dorwart, Chuanjuan Tao, Zengmin Li, Wenjing Guo, Shundi Shi, Daniel Korenblum, Andrew Trans, Anne Aguirre, Edward Liu, Eric T. Harada, James Pollard, Ashwini Bhat, Cynthia Cech, Alexander H. Yang, Cleoma Arnold, Mirkó Palla, Jennifer S. Hovis, Roger B. Chen, Irina Morozova, Sergey M. Kalachikov, James J. Russo, John J. Kasianowicz, Randy Davis, Stefan Roever, George M. Church, Jingyue Ju · 发表于：Proceedings of the National Academy of Sciences · 年份：2016 · DOI：10.1073/pnas.1601782113 · 被引用次数：151 · 研究领域：Nanopore and Nanochannel Transport Studies、Advanced biosensing and bioanalysis techniques、Microfluidic and Capillary Electrophoresis Applications

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 sing...