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

TLDR

Efficient, cost‑effective single‑molecule sequencing platforms enable complete genome decoding, haplotype determination, and detection of alternative splicing. The study demonstrates a nanopore‑based sequencing‑by‑synthesis method that distinguishes DNA bases by detecting distinct polymer tags on the terminal phosphate during incorporation. The approach uses polymer‑tagged nucleotides incorporated by polymerase, releases the tags for detection, and then continues with natural nucleotides, all performed on an electronic chip with an array of independently addressable electrodes each hosting a polymerase–nanopore complex. Nanopore detection shows that polymer tags are far easier to distinguish than natural nucleotides.

Abstract

Significance Efficient cost-effective single-molecule sequencing platforms will facilitate deciphering complete genome sequences, determining haplotypes, and identifying alternatively spliced mRNAs. We demonstrate a single-molecule nanopore-based sequencing by synthesis approach that accurately distinguishes four DNA bases by electronically detecting and differentiating four different polymer tags attached to the terminal phosphate of the nucleotides during their incorporation into a growing DNA strand in the polymerase reaction. With nanopore detection, the distinct polymer tags are much easier to differentiate than natural nucleotides. After tag release, growing DNA chains consist of natural nucleotides allowing long reads. Sequencing is realized on an electronic chip containing an array of independently addressable electrodes, each with a single polymerase–nanopore complex, potentially offering the high throughput required for precision medicine.