Ionic Current-Based Mapping of Short Sequence Motifs in Single DNA Molecules Using Solid-State Nanopores

• Published in: Nano letters Vol. 17; no. 9; pp. 5199 - 5205
• Main Authors: Chen, Kaikai, Juhasz, Matyas, Gularek, Felix, Weinhold, Elmar, Tian, Yu, Keyser, Ulrich F, Bell, Nicholas A. W
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 13.09.2017
• Subjects: Base Sequence; Biotinylation; Chromosome Mapping - methods; DNA - analysis; DNA - genetics; Ion Transport; Letter; Nanopores - ultrastructure; Nanotechnology - methods; Streptavidin - chemistry; single-molecule detection; DNA detection; DNA methyltransferase; S-adenosyl-l-methionine analogue; nanopore sensing; genome mapping
• ISSN: 1530-6984; 1530-6992
• DOI: 10.1021/acs.nanolett.7b01009

Nanopore sensors show great potential for rapid, single-molecule determination of DNA sequence information. Here, we develop an ionic current-based method for determining the positions of short sequence motifs in double-stranded DNA molecules with solid-state nanopores. Using the DNA-methyltransferase M.TaqI and a biotinylated S-adenosyl-l-methionine cofactor analogue we create covalently attached biotin labels at 5′-TCGA-3′ sequence motifs. Monovalent streptavidin is then added to bind to the biotinylated sites giving rise to additional current blockade signals when the DNA passes through a conical quartz nanopore. We determine the relationship between translocation time and position along the DNA contour and find a minimum resolvable distance between two labeled sites of ∼200 bp. We then characterize a variety of DNA molecules by determining the positions of bound streptavidin and show that two short genomes can be simultaneously detected in a mixture. Our method provides a simple, generic single-molecule detection platform enabling DNA characterization in an electrical format suited for portable devices for potential diagnostic applications.