Chemically cleavable 3′-O-allyl-dNTP-allyl-fluorophore fluorescent nucleotide analogues and related methods

• US 9,297,042 B2
• Filed: 08/04/2014
• Issued: 03/29/2016
• Est. Priority Date: 10/31/2005
• 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, Bi, Lanrong, Kim, Dae Hyun, Meng, Qinglin
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/451,265
• Publication Number:
  US 20140377743A1
• Time in Patent Office: 603 Days
• Field of Search: 536/4.1, 536/23.1, 536/24.3, 536/25.3, 536/26.6, 435/6.1, 435/91.1, 422/430
• US Class Current: 1/1
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Abstract

This invention provides a nucleotide analog comprising (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine and uracil, (ii) a deoxyribose, (iii) an allyl moiety bound to the 3′-oxygen of the deoxyribose and (iv) a fluorophore bound to the base via an allyl linker, and methods of nucleic acid sequencing employing the nucleotide analog.

Claims

1. A nucleotide analogue selected from the group consisting of

2. The nucleotide analogue of claim 1, wherein the nucleotide analogue is selected from the group consisting of

3. The nucleotide analogue of claim 1, wherein the fluorophore is selected from the group consisting of ROX, Bodipy-FL-510, Bodipy-650 and R6G.

4. A method for making a nucleotide analogue wherein the nucleotide analogue is selected from the group consisting of

5. The method of claim 4, wherein the steps comprise those set forth in FIG. 2, scheme A;

FIG. 2, scheme B;

FIG. 2, scheme C;

or FIG. 2, scheme D.

6. The method of claim 4, wherein the first suitable solvent is DMF and the second suitable solvent is DMF.

7. The method of claim 4, wherein the first suitable solvent is acetonitrile and the second suitable solvent is DMF.

8. The method of claim 4, wherein the suitable base is NaHCO₃.

9. The method of claim 4, wherein the suitable buffered solvent is DMF buffered with NaHCO₃—

Na₂CO₃.

10. The method of claim 4, wherein in step (a), the N-hydroxysuccinimide ester of a fluorophore contacts 2-(2-amino-ethyl)-prop-2-en-1-ol.

11. The method of claim 10, wherein the steps comprise those set forth in FIG. 6, scheme A;

FIG. 6, scheme B;

FIG. 6, scheme C;

or FIG. 6, scheme D.

19. The method of claim 4, wherein in step (a), the N-hydroxysuccinimide ester of a fluorophore contacts 6-amino-hex-2-en-1-ol.

12. A method for determining the sequence of a DNA comprising performing the following steps for each residue of the DNA to be sequenced:

(a) contacting the DNA with a DNA polymerase in the presence of (i) a primer and (ii) four fluorescent nucleotide analogues under conditions permitting the DNA polymerase to catalyze DNA synthesis,

 

wherein the nucleotide analogues are

13. The method of claim 12, wherein chemically cleaving the fluorophore and the allyl moiety bound to the 3′

-oxygen atom is performed using Na₂PdCl₄.

14. The method of claim 12, wherein about 1000 or fewer copies of the DNA are bound to a solid substrate.

15. The method of claim 12, wherein the four fluorescent nucleotide analogues are selected from the group consisting of

16. The method of claim 12, wherein the DNA polymerase is a 9°

N polymerase.

17. A kit for performing the method of claim 12, comprising, in separate compartments,

(a) a nucleotide analogue selected from the group consisting of

18. The kit of claim 17, wherein the kit comprises