Real-Time DNA Sequencing from Single Polymerase Molecules

TLDR

The study introduces single‑molecule, real‑time sequencing of DNA polymerase using four fluorescently labeled dNTPs. Sequencing is performed by observing fluorescently labeled dNTP incorporation in zero‑mode waveguide arrays, enabling continuous, parallel detection of thousands of polymerase reactions and alignment to a reference to assess polymerization dynamics. The method reveals polymerase dynamics, pause sites, and produces consensus sequences with 99.3 % accuracy at 15× coverage, without systematic errors beyond fluorophore effects.

Abstract

We present single-molecule, real-time sequencing data obtained from a DNA polymerase performing uninterrupted template-directed synthesis using four distinguishable fluorescently labeled deoxyribonucleoside triphosphates (dNTPs). We detected the temporal order of their enzymatic incorporation into a growing DNA strand with zero-mode waveguide nanostructure arrays, which provide optical observation volume confinement and enable parallel, simultaneous detection of thousands of single-molecule sequencing reactions. Conjugation of fluorophores to the terminal phosphate moiety of the dNTPs allows continuous observation of DNA synthesis over thousands of bases without steric hindrance. The data report directly on polymerase dynamics, revealing distinct polymerization states and pause sites corresponding to DNA secondary structure. Sequence data were aligned with the known reference sequence to assay biophysical parameters of polymerization for each template position. Consensus sequences were generated from the single-molecule reads at 15-fold coverage, showing a median accuracy of 99.3%, with no systematic error beyond fluorophore-dependent error rates.