Synthesis of cleavable fluorescent nucleotides as reversible terminators for DNA sequencing by synthesis

• US 10,144,961 B2
• Filed: 05/16/2017
• Issued: 12/04/2018
• Est. Priority Date: 10/19/2007
• 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, Cao, Huanyan, Li, Zengmin, Meng, Qinglin, Guo, Jia, Zhang, Shenglong, Yu, Lin
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US15/596,379
• Publication Number:
  US 20170321267A1
• Time in Patent Office: 567 Days
• Field of Search: 435 61
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/14 Pyrrolo-pyrimidine radicals
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released

Abstract

This invention provides novel azido linkers for deoxynucleotide analogs having a detectable marker attached thereto.

Claims

1. A method for determining the identity of each of a series of consecutive nucleotide residues in a plurality of nucleic acids comprising:

a) contacting the nucleic acids with (i) a composition comprising at least four different dideoxynucleotide triphosphate (ddNTP) analogues, each having the structure;

2. The method of claim 1, wherein the nucleic acid is DNA and the nucleic acid polymerase is a 9°

N thermopolymerase.

3. The method of claim 1, wherein the cleavable linker molecule is an azido cleavable linker molecule or a nitrobenzyl cleavable linker molecule.

4. The method of claim 1, wherein the linker on the ddNTP and the chemical group on the 3′

O position of the dNTP is cleaved with a phosphine.

5. The method of claim 1, wherein the phosphine is charged and water soluble.

6. The method of claim 1, wherein the phosphine is tris (2-carboxyethyl)phosphine.

7. The method of claim 1, wherein the four ddNTP analogues have the following structures:

8. The method of claim 1, wherein the four dNTP analogues have the following structures:

9. The method of claim 7, wherein the four ddNTP analogues have the following structures:

10. The method of claim 1, wherein the cleavable linker molecule in step a) comprises the structure:

11. The method of claim 10, wherein a linker is cleaved by contacting the linker with tris(2-carboxyethyl)phosphine.

12. A method for determining the identity of each of a series of consecutive nucleotide residues in a nucleic acid comprising:

a) contacting the nucleic acid with (i) at least four different deoxynucleotide triphosphate (dNTP) analogues, each having the structure;

13. The method of claim 12, wherein in steps g) and h) the three types of dNTPs are chosen from the group dATP, dCTP, dGTP, dTTP or dITP.

14. The method of claim 12, wherein the cleavable linker molecule in each of step a) and j) independently each comprise the structure:

15. The method of claim 14, wherein a linker is cleaved by contacting the linker with tris(2-carboxyethyl)phosphine.

16. The method of claim 12, wherein the deoxynucleotide analogue in steps g) and h) is a deoxyribonucleotide diphosphate.

17. The method of claim 12, wherein the deoxynucleotide analogue in steps g) and h) is a 3′

-0 blocked deoxynucleotide triphosphate analogue.