Nanopore Based Sequence Specific Detection of Duplex DNA for Genomic Profiling

• Published in: Nano letters Vol. 10; no. 2; pp. 738 - 742
• Main Authors: Singer, Alon, Wanunu, Meni, Morrison, Will, Kuhn, Heiko, Frank-Kamenetskii, Maxim, Meller, Amit
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 10.02.2010
• Subjects: Automatic Data Processing; Automation; Bacteriophage lambda - metabolism; Base Sequence; Condensed matter: structure, mechanical and thermal properties; DNA - chemistry; Exact sciences and technology; Gene Expression Profiling; Genome, Human; Genomics; Humans; Low-dimensional structures (superlattices, quantum well structures, multilayers): structure, and nonelectronic properties; Microscopy, Electron, Transmission - methods; Molecular Sequence Data; Nanoparticles - chemistry; Nanotechnology - methods; Physics; Surfaces and interfaces; thin films and whiskers (structure and nonelectronic properties); PNA; invasion; nanopore; DNA; sequence detection; Amplification; Fluorescent material; Peptides; Genomics; Silicon nitride; Nucleic acids; Nanopore; Nanoporosity
• ISSN: 1530-6984; 1530-6992
• DOI: 10.1021/nl100058y

We demonstrate a purely electrical method for the single-molecule detection of specific DNA sequences, achieved by hybridizing double-stranded DNA (dsDNA) with peptide nucleic acid (PNA) probes and electrophoretically threading the DNA through sub-5 nm silicon nitride pores. Bis-PNAs were used as the tagging probes in order to achieve high affinity and sequence specificity. Sequence detection is performed by reading the ion current traces of individual translocating DNA molecules, which display a characteristic secondary blockade level, absent in untagged molecules. The potential for barcoding DNA is demonstrated through nanopore analysis of once-tagged and twice-tagged DNA at different locations on the same genomic fragment. Our high-throughput, long-read length method can be used to identify key sequences embedded in individual DNA molecules, without the need for amplification or fluorescent/radio labeling. This opens up a wide range of possibilities in human genomics as well as in pathogen detection for fighting infectious diseases.