DNA sequencing by synthesis using modified nucleotides and nanopore detection

Patent metadata

Field	Value
Publication number	US-10443096-B2
Application number	US-201113994431-A
Country	US
Kind code	B2
Filing date	Dec 16, 2011
Priority date	Dec 17, 2010
Publication date	Oct 15, 2019
Grant date	Oct 15, 2019

Abstract

This disclosure is related to a method of sequencing a single-stranded DNA using deoxynucleotide polyphosphate analogues and translocation of tags from incorporated deoxynucleotide polyphosphate analogues through a nanopore.

First claim

What is claimed is:

1. A method for sequencing a nucleic acid molecule, the method comprising:
a) providing a chip comprising a plurality of nanopores, wherein said plurality of nanopores comprises a nanopore in a membrane adjacent to an electrode, wherein said nanopore is linked to a nucleic acid polymerase;
b) providing at least one tagged nucleotide polyphosphate (NPP) analogue, wherein the tagged NPP comprises a tag capable of being released during polymerization catalyzed by said nucleic acid polymerase and detected with the aid of said nanopore, and wherein said tag comprises an oligonucleotide;
c) contacting said at least one tagged NPP and the nucleic acid molecule with said nucleic acid polymerase linked to said nanopore, whereby said polymerase polymerizes said tagged NPP into a strand that is complementary to at least a portion of said nucleic acid molecule, wherein during polymerization said tag is released from said tagged nucleotide, and wherein said released tag flows through said nanopore;
d) detecting the released tag with the aid of said electrode, wherein the tag is detected subsequent to being released from said tagged nucleotide; and
e) correlating said tag with a type of tagged NPP, and generating, a nucleic acid sequence of the nucleic acid molecule based upon said detected tag.

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

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

4. The method of claim 1 , wherein the nanopore is an alpha-hemolysin nanopore.

5. The method of claim 1 , wherein:
(I) in step (a) the chip, nanopore, and nucleic acid are in an electrolyte solution;
(II) the tagged nucleotide has a polyphosphate portion having a tag attached thereto, wherein during polymerization the tag is released from the tagged nucleotide;
(III) in step (c) a voltage is applied across the membrane; and
(IV) in step (d) measuring an electronic change across the nanopore over time resulting from a current blockage caused by the tag flowing through the nanopore, thereby identifying the nucleotide residue in the nucleic acid complementary to the incorporated tagged nucleotide

thereby determining the nucleotide sequence of the nucleic acid.

6. The method of claim 5 :
wherein the nucleic acid molecule is a single-stranded nucleic acid molecule;
wherein the single-stranded nucleic acid molecule is contacted in step (c) by at least four NPP analogues; and
wherein (i) the type of base on each NPP analogue is different from the type of base on each of the other three NPP analogues, and (ii) either the number of phosphates in the polyphosphate portion of each NPP analogue is different from the number of phosphates in the polyphosphate portion of each of the other three NPP analogues, or the number of phosphates in the polyphosphate portion of each of the four NPP analogues is the same and the type of tag on each NPP analogue is different from the type of tag on each of the other three NPP analogues.

7. The method of claim 5 , wherein each NPP is a dNPP.

8. The method of claim 5 :
(i) wherein in step (c) the nucleic acid is contacted with at least four nucleotide polyphosphate (NPP) analogues wherein each of the four NPP analogues has the structure:

 wherein the base is adenine, guanine, cytosine, thymine or uracil, or a derivative of each thereof, wherein R 1 is OH, wherein R 2 is H or OH, wherein X is a linker which links the terminal phosphate to the tag, wherein n is 1, 2, 3, or 4, wherein Z is O, S, or BH 3 , and with the proviso that (i) the type of base on each NPP analogue is different from the type of base on each of the other three NPP analogues, and (ii) either the value of n of each NPP analogue is different from the value of n of each of the other three NPP analogues, or the value of n of each of the four NPP analogues is the same and the type of tag on each NPP analogue is different from the type of tag on each of the other three NPP analogues;
(ii) wherein in step (d) the electronic change is different for each value of n, or for each different type of tag, as appropriate, so as to thereby identify the nucleotide residue in the nucleic acid complementary to the incorporated NPP analogue; and
(iii) further, iteratively performing steps (a) through (e) for each nucleotide residue of the nucleic acid being sequenced, wherein in each iteration of step (c) the NPP analogue is incorporated into the nucleic acid extension product resulting from the previous iteration of step (c) if it is complementary to the nucleotide residue of the nucleic acid which is immediately 5′ to a nucleotide residue of the nucleic acid hybridized to the 3′ terminal nucleotide residue of the nucleic acid extension product,
thereby determining the nucleotide sequence of the nucleic acid.

9. The method of claim 8 , wherein the tag further comprises one or more of a polyethylene glycol, an amino acid, a carbohydrate, a dye, a mononucleotide, a dinucleotide, a trinucleotide, a tetranucleotide, a pentanucleotide or a hexanucleotide, a fluorescent dye, a chemiluminiscent compound, a peptide, a nucleotide monophosphate, a nucleotide diphosphate, an aliphatic acid or an aromatic acid or an alcohol or a thiol with unsubstituted or substituted with one or more halogens, a cyano group, a nitro group, an alkyl group, an alkenyl group, an alkynyl group, or an azido group.

10. The method of claim 8 , wherein the base is selected from the group consisting of adenine, guanine, cytosine, thymine, 7-deazaguanine, 7-deazaadenine or 5-methylcytosine.

11. The method of claim 8 , further comprising a washing step after each iteration of step (c) to remove unincorporated NPP analogues from contact with the nucleic acid.

12. The method of claim 8 , wherein the nucleic acid, electrolyte solution and nanopore in the membrane are located within a single container.

13. The method of claim 8 , wherein the NPP analogues have the following structures:

wherein R 1 is OH, wherein R 2 is H or OH, wherein Z is O, S, or BH 3 , and wherein the base is adenine, guanine, cytosine, thymine, uracil, a 7-deazapurine or a 5-methylpyrimidine.

14. The method of claim 8 , wherein the bases of the oligonucleotide of the tag are the same type of base as the base of the NPP analogue.

15. The method of claim 8 , wherein the NPP analogues are chosen from the following:

wherein in each structure n is, independently, 1, 2, 3 or 4, and m is, independently, an integer from 0 to 100, and wherein when m is 0 the terminal phosphate of the NPP is bonded directly to the 3′ O atom of the nucleoside shown on the left hand side of the structure, and wherein the value of n is different for each type of base.

16. The method of claim 8 , wherein the electronic change is a change in current amplitude.

17. The method of claim 8 , wherein the electronic change is a change in conductance of the nanopore.

18. The method of claim 8 , wherein the nanopore is biological.

19. The method of claim 18 , wherein the nanopore is proteinaceous.

20. Th

Assignees

• Ju Jingyue
• Kumar Shiv
• Li Zengmin
• Tao Chuanjuan
• Chien Minchen
• Russo James J
• Kalachikov Sergey
• Shepard Ken
• Rosenstein Jacob Karl
• Univ Columbia

Inventors

• Ju Jingyue
• Kumar Shiv
• Li Zengmin
• Tao Chuanjuan
• Chien Minchen
• Russo James J
• Kalachikov Sergey
• Shepard Ken
• Rosenstein Jacob Karl

Classifications

• C07H21/00

  Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids · CPC title

• C07H19/10

  with the saccharide radical esterified by phosphoric or polyphosphoric acids · CPC title

• G01N33/48721

  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) · CPC title

• C07H21/02

  with ribosyl as saccharide radical · CPC title

• C07H1/00

  Processes for the preparation of sugar derivatives · CPC title