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Design and characterization of a nanopore-coupled polymerase for single-molecule DNA sequencing by synthesis on an electrode array
P.B. Stranges, M. Palla, S. Kalachikov, J. Nivala, M. Dorwart, A. Trans, S. Kumar, , M. Chien, C. TaoShow More
Published in National Academy of Sciences
2016
PMID: 27729524
Volume: 113
   
Issue: 44
Pages: E6749 - E6756
Abstract
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 a-hemolysin (aHL) 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.
About the journal
JournalProceedings of the National Academy of Sciences of the United States of America
PublisherNational Academy of Sciences
ISSN00278424
Open AccessNo