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

TLDR

DNA sequencing is rapidly expanding in life science research and clinical medicine, and polymer‑tagged nucleotides have been shown to serve as viable substrates for replication that can be electronically detected in a nanopore. The study aims to design and characterize a DNA polymerase–nanopore protein construct integrated on a chip, establishing a foundation for a scalable, single‑molecule, electronic DNA‑sequencing platform. The construct incorporates all four tagged nucleotides and employs real‑time detection of single‑tagged‑nucleotide addition via a nanopore coupled to an electrode array.

Abstract

Significance DNA sequencing has been dramatically expanding its scope in basic life science research and clinical medicine. Recently, a set of polymer-tagged nucleotides were shown to be viable substrates for replication and electronically detectable in a nanopore. Here, we describe the design and characterization of a DNA polymerase–nanopore protein construct on an integrated chip. This system incorporates all four tagged nucleotides and distinguishes single–tagged-nucleotide addition in real time. Coupling protein catalysis and nanopore-based detection to an electrode array could provide the foundation of a highly scalable, single-molecule, electronic DNA-sequencing platform.