# US 10,246,479 B2 · Method of preparation of nanopore and uses thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,246,479 B2 - Document ID: US10246479B2 - Status: Granted - Kind: B2 - Application number: 14/391,320 - Filed: 2013-04-08 - Published: 2015-04-23 - Granted: 2019-04-02 - Earliest claimed: 2012-04-09 - Assignees: The Trustees of Columbia University in the City of New York; The United States of America as represented by the Secretary of Commerce - Applicants: Jingyue Ju; Shiv Kumar; Chuanjuan Tao; Minchen Chien; James J. Russo; John J. Kasianowicz; Joseph W. F. Robertson - Inventors: Jingyue Ju; Shiv Kumar; Chuanjuan Tao; Minchen Chien; James J. Russo; John J. Kasianowicz; Joseph W. F. Robertson - Web page: https://patentgrep.com/patent/US10246479B2 ## CPC - C07H 19/00: Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides ; Anhydro-derivatives thereof - C07H 19/06: Pyrimidine radicals - C07H 19/16: Purine radicals - C12Q 1/42: involving phosphatase - C12Q 1/48: involving transferase - C12Q 1/6869: Methods for sequencing - C12Q 1/6883: for diseases caused by alterations of genetic material - G01N 33/48721: {Investigating individual macromolecules, e.g. by translocation through nanopores (Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68)} - B82Y 5/00: Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery - C12Q 2600/158: Expression markers - G01N 2333/9125: with a definite EC number (2.7.7.-) - C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES ## Abstract This disclosure provides systems and methods for sequencing nucleic acids using nucleotide analogues and translocation of tags from incorporated nucleotide analogues through a nanopore. In aspects, this disclosure is related to composition, method, and system for sequencing a nucleic acid using tag molecules and detection of translocation through a nanopore of tags released from incorporation of the molecule. ## Claims ### Claim 1 A tagged nucleotide, wherein the nucleotide comprises a tag capable of being cleaved in a nucleotide polymerization event and detected with the aid of a nanopore, wherein the tagged nucleotide has the structure: wherein the tag comprises an oligonucleotide which comprises nucleotides having natural bases and (i) at least one abasic site or (ii) at least one nucleotide having a modified base; wherein R1 is OH, wherein R2 is H or OH, wherein X is a linker and may comprise O, NH, S, or CH2, wherein Z is O, S, or BH3, wherein the base is adenine, guanine, cytosine, thymine, uracil, or a derivative of one of these bases, wherein n is 1, 2, 3, or 4. ### Claim 2 (depends on 1) The tagged nucleotide of claim 1, wherein the tag does not comprise a fluorophore. ### Claim 3 (depends on 1) The tagged nucleotide of claim 1, wherein the tag is detectable by its charge, shape, size, or any combination thereof. ### Claim 4 (depends on 1) The tagged nucleotide of claim 1, wherein the tag comprises an additional identifiable moiety. ### Claim 5 (depends on 4) The tagged nucleotide of claim 4, wherein the additional identifiable moiety is a coumarin-based dye. ### Claim 6 (depends on 1) A composition comprising four different types of the tagged nucleotide of claim 1, wherein the base of a first type of tagged nucleotide is adenine or a derivative thereof, the base of a second type of tagged nucleotide is guanine or a derivative thereof, the base of a third type of tagged nucleotide is cytosine or a derivative thereof, and the base of a fourth type of tagged nucleotide is thymine or a derivative thereof or uracil or a derivative thereof, and wherein the tag on each type of tagged nucleotide is different from the tag on each of the other three types of tagged nucleotide. ### Claim 7 (depends on 1) The tagged nucleotide of claim 1, wherein the tag further comprises one or more of ethylene glycol, an amino acid, a carbohydrate, a peptide, a dye, a chemiluminescent compound, a mononucleotide, a dinucleotide, a trinucleotide, a tetranucleotide, a pentanucleotide, a hexanucleotide, an aliphatic acid, an aromatic acid, an alcohol, a thiol group, a cyano group, a nitro group, an alkyl group, an alkenyl group, an alkynyl group, an azido group, or a combination thereof. ### Claim 8 (depends on 1) The tagged nucleotide of claim 1, wherein the oligonucleotide has a length of approximately 30 bases. ### Claim 9 (depends on 1) The tagged nucleotide of claim 1, wherein n is four. ### Claim 10 (depends on 1) The tagged nucleotide of claim 1, wherein the oligonucleotide comprises a 5′-OH, and the 5′-OH of said oligonucleotide is attached to the terminal phosphate of the tagged nucleotide analogue. ### Claim 11 (depends on 1) The tagged nucleotide of claim 1, wherein the oligonucleotide comprises at least one abasic site. ### Claim 12 (depends on 1) The tagged nucleotide of claim 1, wherein the oligonucleotide comprises at least one nucleotide having a modified base. ### Claim 13 (depends on 1) The tagged nucleotide of claim 1, wherein the tag has a charge which is reverse in sign relative to the charge on the rest of the tagged nucleotide. ### Claim 14 (depends on 13) The tagged nucleotide of claim 13, wherein the magnitude of the charge on the tag is the same as the magnitude of the charge on the remainder of the tagged nucleotide. ### Claim 15 (depends on 13) The tagged nucleotide of claim 13, wherein the tag has a positive charge. ### Claim 16 (depends on 13) The tagged nucleotide of claim 13, wherein the tag further comprises lysines or arginines having a positive charge, wherein the positive charge of the lysines or arginines neutralizes the negative charge of the phosphates of the tagged nucleotide. ### Claim 17 (depends on 13) The tagged nucleotide of claim 13, wherein removal of the tag changes the charge of the tagged nucleotide.