Method of preparation of nanopore and uses thereof

• US 10,246,479 B2
• Filed: 04/08/2013
• Issued: 04/02/2019
• Est. Priority Date: 04/09/2012
• Status: Active Grant

• Current Assignee: THE GOVERNMENT OF THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY OF COMMERCE
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University), THE GOVERNMENT OF THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY OF COMMERCE
• Inventors: Ju, Jingyue, Kumar, Shiv, Tao, Chuanjuan, Chien, Minchen, Russo, James J., Kasianowicz, John J., Robertson, Joseph W. F.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/391,320
• Publication Number:
  US 20150111759A1
• Time in Patent Office: 2,185 Days
• Field of Search: 435 61, 536 41
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07H 19/00 Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2521/525 Phosphatase
• CPC Class Codes: C12Q 2521/543 Immobilised enzyme(s)
• CPC Class Codes: C12Q 2535/107 Maxam and Gilbert method, i.e. sequential release and detection of nucleotides
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 2333/9125 with a definite EC number (2.7.7.-)
• CPC Class Codes: 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

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

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:

2. The tagged nucleotide of claim 1, wherein the tag does not comprise a fluorophore.

3. The tagged nucleotide of claim 1, wherein the tag is detectable by its charge, shape, size, or any combination thereof.

4. The tagged nucleotide of claim 1, wherein the tag comprises an additional identifiable moiety.

5. The tagged nucleotide of claim 4, wherein the additional identifiable moiety is a coumarin-based dye.

6. 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.

7. 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.

8. The tagged nucleotide of claim 1, wherein the oligonucleotide has a length of approximately 30 bases.

9. The tagged nucleotide of claim 1, wherein n is four.

10. 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.

11. The tagged nucleotide of claim 1, wherein the oligonucleotide comprises at least one abasic site.

12. The tagged nucleotide of claim 1, wherein the oligonucleotide comprises at least one nucleotide having a modified base.

13. 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.

14. 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.

15. The tagged nucleotide of claim 13, wherein the tag has a positive charge.

16. 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.

17. The tagged nucleotide of claim 13, wherein removal of the tag changes the charge of the tagged nucleotide.