Nanopore based sequence specific detection of duplex DNA for genomic profiling.
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.
Singer
Alon
A
Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, USA.
Wanunu
Meni
M
Morrison
Will
W
Kuhn
Heiko
H
Frank-Kamenetskii
Maxim
M
Meller
Amit
A
D001331:Automation; D010582:Bacteriophage lambda / Q000378:metabolism; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D001330:Electronic Data Processing; D020869:Gene Expression Profiling; D015894:Genome, Human; D023281:Genomics; D006801:Humans; D046529:Microscopy, Electron, Transmission / Q000379:methods; D008969:Molecular Sequence Data; D053758:Nanoparticles / Q000737:chemistry*; D036103:Nanotechnology / Q000379:methods*
D004247:DNA