In Situ Unzipping of Long, High Molecular Weight DNA in Solid State Nanopores

Abstract Nanopores are an established paradigm in genome sequencing technology, with remarkable advances still being made today. All efforts continually address the challenges associated with rapid, accurate, high-throughput, and low cost detection, particularly with long-read length DNA. We report on the in situ melting and unzipping of long, high molecular weight DNA. At varying salt concentration, we directly compare the translocation conductance and speeds between SiN and graphene nanopores at sub-10 nm pore diameters. We observe the force-induced unzipping of dsDNA at higher salt concentrations than previously reported in literature. We observe free running translocation without secondary structures of ssDNA that is an order of magnitude longer than reported before. We hypothesize that the frayed single strands at the molecule’s end get captured with a higher likelihood than both ends together. In understanding this phenomena for long-read lengths, we continue to address the challenges revolving around future generations of sequencing technology. Statement of SignificanceGenome sequencing is an advancing field with applications in clinical diagnostics. However, the challenges of providing accurate identification of longer DNA molecules at low cost are still developing. While detection of long DNA molecules is established, the identification of its individual nucleotides presents its own set of challenges. By separating the hydrogen bonds between the two strands, individual nucleotides are made directly measurable. However, identification is hindered from the formation of secondary structures, where the single-stranded DNA sticks to itself. Previous studies only included short DNA molecules. We report in situ force-induced unzipping and translocation of long DNA without secondary structures almost an order of magnitude longer than reported before. Our findings present new experimental conditions and insights that progress the field towards high accuracy sequencing of individual long molecules.