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

نویسندگان

  • P Benjamin Stranges
  • Mirkó Palla
  • Sergey Kalachikov
  • Jeff Nivala
  • Michael Dorwart
  • Andrew Trans
  • Shiv Kumar
  • Mintu Porel
  • Minchen Chien
  • Chuanjuan Tao
  • Irina Morozova
  • Zengmin Li
  • Shundi Shi
  • Aman Aberra
  • Cleoma Arnold
  • Alexander Yang
  • Anne Aguirre
  • Eric T Harada
  • Daniel Korenblum
  • James Pollard
  • Ashwini Bhat
  • Dmitriy Gremyachinskiy
  • Arek Bibillo
  • Roger Chen
  • Randy Davis
  • James J Russo
  • Carl W Fuller
  • Stefan Roever
  • Jingyue Ju
  • George M Church
چکیده

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.

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عنوان ژورنال:
  • Proceedings of the National Academy of Sciences of the United States of America

دوره 113 44  شماره 

صفحات  -

تاریخ انتشار 2016