Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators

• US 10,000,801 B2
• Filed: 11/17/2016
• Issued: 06/19/2018
• Est. Priority Date: 12/01/2006
• Status: Active Grant

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Ju, Jingyue, Kim, Dae Hyun, Bi, Lanrong, Meng, Qinglin, Li, Xiaoxu
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US15/354,531
• Publication Number:
  US 20170114403A1
• Time in Patent Office: 579 Days
• Field of Search: 435 61, 435 911, 435 912, 536 41, 536 231, 536 243, 536 266
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore

Abstract

This invention provides a process for sequencing single-stranded DNA employing modified nucleotides.

Claims

1. A method for determining the sequence of DNA covalently immobilized in a plurality of spots on a solid surface, wherein each spot comprises a plurality of DNAs to be sequenced comprising the same series of consecutive residues, said method comprising performing the following steps for each residue of the plurality of DNAs to be sequenced:

(a) contacting the plurality of DNAs to be sequenced with a DNA polymerase, a primer, and four nucleotide analogues, under conditions permitting the DNA polymerase to catalyze DNA synthesis, wherein,

the four nucleotide analogues;

(1) consist of an analogue of dGTP, an analogue of dCTP, an analogue of dTTP or dUTP, and an analogue of dATP,

(2) each comprise (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine or uracil, and analogues thereof, (ii) a deoxyribose, (iii) a moiety cleavably linked to the 3′

-oxygen of the deoxyribose and (iv) a unique label cleavably linked to the base, so that a nucleotide analogue complementary to the residue-being sequenced is incorporated by the DNA polymerase into a DNA of the plurality of DNAs to be sequenced, and,

(3) each of the four nucleotide analogues has a unique label which is different than the unique labels of the other three analogues;

(b) removing unbound nucleotide analogues;

(c) contacting the plurality of DNAs to be sequenced with four reversible terminators in the presence of a DNA polymerase and a primer, under conditions permitting the DNA polymerase to catalyze DNA synthesis, wherein

the four reversible terminators;

(1) consist of an analogue of dGTP, an analogue of dCTP, an analogue of dTTP or dUTP, and an analogue of dATP, and

(2) each comprise (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine or uracil, and analogues thereof, which base does not have a unique label bound thereto, (ii) a deoxyribose, and (iii) a moiety cleavably linked to the 3′

-oxygen of the deoxyribose;

(d) removing unbound reversible terminators;

(e) determining the identity of the nucleotide analogues incorporated in step (a) via determining the identity of the corresponding unique label, with the proviso that step (e) can either precede step (c) or follow step (d); and

(f) following step (e), except with respect to the final DNA residue of the plurality of DNAs to be sequenced, cleaving from the incorporated nucleotide analogues the unique label, if applicable, and the moiety linked to the 3′

-oxygen atom of the deoxyribose of the incorporated nucleotide analogues and/or reversible terminators,

thereby determining the sequence of the plurality of DNAs being sequenced.

2. A kit for performing the method of claim 1, comprising, in separate compartments,

(a) nucleotide analogues of (i) GTP, (ii) ATP, (iii) CTP and (iv) TTP or UTP, wherein each nucleotide analogue comprises (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine or uracil, or an analogue thereof, (ii) a deoxyribose, (iii) a cleavable moiety bound to the 3′

-oxygen of the deoxyribose and (iv) a unique label bound to the base via a cleavable linker,

(b) reversible terminators comprising an analogue of (i) GTP, (ii) ATP, (iii) CTP and (iv) TTP or UTP, wherein each reversible terminator comprises (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine or uracil, or an analogue thereof, which base does not have a unique label bound thereto, (ii) a deoxyribose, and (iii) a cleavable moiety bound to the 3′

-oxygen of the deoxyribose;

(c) reagents suitable for use in DNA polymerization; and

(d) instructions for use.

3. The kit of claim 2, wherein the nucleotide analogues of part (a) are 3′

-O-allyl-dGTP-allyl-Cy5, 3′

-O-allyl-dCTP-allyl-Bodipy-FL-510, 3′

-O-allyl-dATP- allyl-ROX and 3′

-O-allyl-dUTP-allyl-R6G.

4. The kit of claim 2, wherein the nucleotide analogues of part (a) are 3′

-O-allyl-dGTP-allyl-Bodipy-FL-510, 3′

-O-allyl-dCTP-allyl-Bodipy-650, 3′

-O-allyl-dATP-allyl-ROX and 3′

-O-allyl-dUTP-allyl-R6G.

5. The kit of claim 2, wherein the nucleotide analogues of part (a) are 3′

-O-allyl-dGTP-allyl-Bodipy-650, 3′

-O-allyl-dCTP-allyl-Bodipy-FL-510, 3′

-O-allyl-dATP-allyl-ROX and 3′

-O-allyl-dUTP-allyl-R6G.

6. The kit of claim 2, wherein the reversible terminators of part (b) are 3′

-O-allyl-dGTP, 3′

-O-allyl-dCTP, 3′

-O-allyl-dATP and 3′

-O-allyl-dUTP.

7. The kit of claim 2, wherein the reversible terminators in part b) are 3′

-O-2-nitrobenzyl-dGTP, 3′

-O-2-nitrobenzyl-dCTP, 3′

-O-2-nitrobenzyl-dATP and 3′

-O-2-nitrobenzyl-dUTP.

8. The method of claim 1, wherein the moiety cleavably linked to the 3′

-oxygen of the deoxyribose of each of the four nucleotide analogues and/or each of the four reversible terminators is chemically cleavable or photocleavable.

9. The method of claim 1, wherein the moiety cleavably linked to the 3′

-oxygen of the deoxyribose in each of the four nucleotide analogues and/or each of the four reversible terminators is an allyl moiety or a 2-nitrobenzyl moiety.

10. The method of claim 1, wherein the unique label bound to the base of each of the four nucleotide analogues via a cleavable linker is a dye, a fluorophore, a chromophore, a combinatorial fluorescence energy transfer tag, a mass tag, or an electrophore.

11. A method for determining the sequence of DNA covalently immobilized in a plurality of spots on a solid surface, wherein each spot comprises a plurality of DNAs to be sequenced comprising the same series of consecutive residues, said method comprising performing the following steps for each residue of the plurality of DNAs to be sequenced:

(a) contacting the plurality of DNAs to be sequenced with a DNA polymerase, a primer, four nucleotide analogues, under conditions permitting the DNA polymerase to catalyze DNA synthesis, wherein

the four nucleotide analogues;

(1) consist of an analogue of dGTP, an analogue of dCTP, an analogue of dTTP or dUTP, and an analogue of dATP,

(2) comprise (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine or uracil, and analogues thereof, (ii) a deoxyribose, (iii) a moiety cleavably linked to the 3′

-oxygen of the deoxyribose and (iv) a unique label cleavably linked to the base, so that a nucleotide analogue complementary to the residue being sequenced is incorporated by the DNA polymerase into a DNA of the plurality of DNAs to be sequenced, and

(3) each have a unique label which is different than the unique labels of the other three nucleotide analogues, and

subsequently contacting the plurality of DNAs to be sequenced with four reversible terminators under conditions permitting the DNA polymerase to catalyze DNA synthesis, wherein;

the four reversible terminators;

(1) consist of an analogue of dGTP, an analogue of dCTP, an analogue of dTTP or dUTP, and an analogue of dATP, and

(2) each comprise (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine or uracil, and analogues thereof, which base does not have a unique label bound thereto, (ii) a deoxyribose, and (iii) a moiety cleavably linked to the 3′

-oxygen of the deoxyribose;

(b) removing unbound reversible terminators and removing unbound nucleotide analogues;

(c) determining the identity of the nucleotide analogues incorporated in step (a) via determining the identity of the corresponding unique label; and

(d) following step (c), except with respect to the final DNA residue of the plurality of DNAs to be sequenced, cleaving from the incorporated nucleotide analogues the unique label, if applicable, and the moiety linked to the 3′

-oxygen atom of the deoxyribose of the incorporated nucleotide analogues and/or reversible terminators,

thereby determining the sequence of the plurality of DNAs being sequenced.

12. The method of claim 11, wherein the moiety cleavably linked to the 3′

-oxygen of the deoxyribose of each of the four nucleotide analogues and/or each of the four reversible terminators is chemically cleavable or photocleavable.

13. The method of claim 11, wherein the moiety cleavably linked to the 3′

-oxygen of the deoxyribose of each of the four nucleotide analogues and/or each of the four reversible terminators is an allyl moiety or a 2-nitrobenzyl moiety.

14. The method of claim 11, wherein the unique label bound to the base of each of the four nucleotide analogues via a cleavable linker is a dye, a fluorophore, a chromophore, a combinatorial fluorescence energy transfer tag, a mass tag, or an electrophore.

15. A kit for performing the method of claim 11, comprising, in separate compartments,

(a) nucleotide analogues of (i) GTP, (ii) ATP, (iii) CTP and (iv) TTP or UTP, wherein each nucleotide analogue comprises (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine or uracil, or an analogue thereof, (ii) a deoxyribose, (iii) a cleavable moiety bound to the 3′

-oxygen of the deoxyribose and (iv) a unique label bound to the base via a cleavable linker,

(b) reversible terminators comprising an analogue of (i) GTP, (ii) ATP, (iii) CTP and (iv) TTP or UTP, wherein each reversible terminator comprises (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine or uracil, or an analogue thereof, which base does not have a unique label bound thereto, (ii) a deoxyribose, and (iii) a cleavable moiety bound to the 3′

-oxygen of the deoxyribose;

(c) reagents suitable for use in DNA polymerization; and

(d) instructions for use.

16. The kit of claim 15, wherein the four nucleotide analogues of part (a) are 3′

-O-allyl-dGTP-allyl-Cy5, 3′

-O-allyl-dCTP-allyl-Bodipy-FL-510, 3′

-O-allyl-dATP-allyl-ROX and 3′

-O-allyl-dUTP-allyl-R6G.

17. The kit of claim 15, wherein the four nucleotide analogues of part (a) are 3′

-O-allyl-dGTP-allyl-Bodipy-FL-510, 3′

-O-allyl-dCTP-allyl-Bodipy-650, 3′

-O-allyl-dATP-allyl-ROX and 3′

-O-allyl-dUTP-allyl-R6G.

18. The kit of claim 15, wherein the four nucleotide analogues of part (a) are 3′

-O-allyl-dGTP-allyl-Bodipy-650, 3′

-O-allyl-dCTP-allyl-Bodipy-FL-510, 3′

-O-allyl-dATP-allyl-ROX and 3′

-O-allyl-dUTP-allyl-R6G.

19. The kit of claim 15, wherein the four reversible terminators of part (b) are 3′

-O-allyl-dGTP, 3′

-O-allyl-dCTP, 3′

-O-allyl-dATP and 3′

-O-allyl-dUTP.

20. The kit of claim 15, wherein the four reversible terminators in part (b) are 3′

-O-2-nitrobenzyl-dGTP, 3′

-O-2-nitrobenzyl-dCTP, 3′

-O-2-nitrobenzyl-dATP and 3′

-O-2-nitrobenzyl-dUTP.