Towards Dark Quencher Based Real Time DNA Sequencing

Abstract

Third-generation DNA sequencing technologies are expected to transform biomedical research and health care. Although powerful single-molecule DNA sequencing methods are available, they suffer from significant limitations, including the need for sophisticated instrumentation, microfluidics, or nanofabrication. Here, we introduce a simpler approach based on fluorescently labelled DNA polymerases and dark quencher labelled nucleotides (dNPP-Q, penta-phosphates). During the time between the binding and incorporation of a nucleotide, the fluorescence intensity of the polymerase-attached fluorophores is differentially reduced due to Foerster resonance energy transfer to a nucleotide-specific dark-quencher attached to the terminal phosphate group. Use of dark-quenchers enables real-time sequencing with long read lengths at micromolar nucleotide concentrations, and is compatible with standard total-internal-reflection fluorescence microscopy.We first characterized dark quenchers for single-molecule detection by studying a set of double-stranded DNA constructs labelled with two fluorophores (“green” and “red”) and one dark-quencher. We then designed a biochemical system that allowed us to observe directly individual events of dNPP-Q binding to a binary complex of a DNA polymerase (labelled with a green fluorophore) bound to a primer-template substrate labelled with a red fluorophore. Binding events of dNPP-Q to the complex results in real-time observations of clear, transient reductions of green and red fluorescence by 80% and 30%, respectively; the quenching efficiency matches to the expectations based on the photophysical properties of the interacting chromophores and their separation. We are currently extending the concept to the remaining bases. Our approach should facilitate the study of DNA- and RNA-polymerase mechanisms as well as the development of faster and cheaper methods for single-molecule DNA sequencing.