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

• US 9,175,342 B2
• Filed: 07/25/2013
• Issued: 11/03/2015
• 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
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/951,269
• Publication Number:
  US 20140093869A1
• Time in Patent Office: 831 Days
• Field of Search: 435/6.1, 435/91.1, 435/91.2, 536/4.1, 536/23.1, 536/26.6
• US Class Current: 1/1
• 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 analogues having a detectable marker attached thereto.

Claims

1. A compound having a first, a second, and a third portion, wherein the second portion has the following structure:

2. The compound of claim 1, wherein the detectable marker is a fluorescent dye.

3. The compound of claim 1, wherein the first portion is a deoxynucleotide comprising a methylazido group attached to a 3′

O atom thereof.

4. The compound of claim 2, having the structure:

5. The compound of claim 4 having the structure:

6. An array comprising a nucleic acid attached to a solid surface, wherein the nucleic acid comprises a methylazido group attached to a 3′

O atom thereof and a molecule having the structure;

7. 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;

8. The method of claim 7, wherein a linker is cleaved by contacting the linker with tris(2-carboxyethyl)phosphine.

9. The method of claim 7, wherein one or more linkers are photocleavable or chemically cleavable.

10. The method of claim 7, wherein one or more chemical groups are photocleavable or chemically cleavable.

11. The method of claim 7, wherein R in the structures set forth in steps a) and/or j) is independently chosen from a —

N₃ group or an allyl group.

12. The method of claim 7, wherein the cleavable chemical group in step g) is independently chosen from a —

N₃ group or an allyl group.