title: PEG-labeled nucleotides and nanopore detection for single molecule DNA sequencing by synthesis.
abstract: We describe a novel single molecule nanopore-based sequencing by synthesis (Nano-SBS) strategy that can accurately distinguish four bases by detecting 4 different sized tags released from 5'-phosphate-modified nucleotides. The basic principle is as follows. As each nucleotide is incorporated into the growing DNA strand during the polymerase reaction, its tag is released and enters a nanopore in release order. This produces a unique ionic current blockade signature due to the tag's distinct chemical structure, thereby determining DNA sequence electronically at single molecule level with single base resolution. As proof of principle, we attached four different length PEG-coumarin tags to the terminal phosphate of 2'-deoxyguanosine-5'-tetraphosphate. We demonstrate efficient, accurate incorporation of the nucleotide analogs during the polymerase reaction, and excellent discrimination among the four tags based on nanopore ionic currents. This approach coupled with polymerase attached to the nanopores in an array format should yield a single-molecule electronic Nano-SBS platform.
authors.affiliation: Center for Genome Technology & Biomolecular Engineering, Department of Chemical Engineering, Columbia University, New York, NY 10027, USA.
authors.forename: Shiv
authors.initials: S
authors.lastname: Kumar
authors.forename: Chuanjuan
authors.initials: C
authors.lastname: Tao
authors.forename: Minchen
authors.initials: M
authors.lastname: Chien
authors.forename: Brittney
authors.initials: B
authors.lastname: Hellner
authors.forename: Arvind
authors.initials: A
authors.lastname: Balijepalli
authors.forename: Joseph W F
authors.initials: JW
authors.lastname: Robertson
authors.forename: Zengmin
authors.initials: Z
authors.lastname: Li
authors.forename: James J
authors.initials: JJ
authors.lastname: Russo
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Reiner
authors.forename: John J
authors.initials: JJ
authors.lastname: Kasianowicz
authors.forename: Jingyue
authors.initials: J
authors.lastname: Ju
mesh_terms: D003374:Coumarins / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D003848:Deoxyguanine Nucleotides / Q000032:analysis* / Q000737:chemistry; D004560:Electricity; D055664:Electrochemical Techniques / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes; D008970:Molecular Weight; D058608:Nanopores; D009711:Nucleotides / Q000032:analysis* / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D003374:Coumarins; D003848:Deoxyguanine Nucleotides; D005456:Fluorescent Dyes; D009711:Nucleotides; D011092:Polyethylene Glycols; D004247:DNA; C030123:coumarin