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

Patent metadata

Field	Value
Publication number	US-9297042-B2
Application number	US-201414451265-A
Country	US
Kind code	B2
Filing date	Aug 4, 2014
Priority date	Oct 31, 2005
Publication date	Mar 29, 2016
Grant date	Mar 29, 2016

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.

First claim

What is claimed is:

1. A nucleotide analogue selected from the group consisting of

wherein F comprises a fluorophore.

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

comprising the steps of:
(a) contacting an N-hydroxysuccinimide ester of a fluorophore and either 6-amino-hex-2-en-1-ol or 2-(2-amino-ethyl)-prop-2-en-1-ol in the presence of a first suitable solvent and a suitable base in the presence of a first suitable solvent and a suitable base;
(b) treating the resulting product of step (a) with DSC/Et 3 N in a second suitable solvent; and
(c) treating the resulting product of step (b) with a 3′-O-allyl-dNTP-NH 2 in the presence of a suitable buffered solvent, wherein the base of the 3′-O-allyl-dNTP-NH 2 is an adenine, guanine, cytosine, uracil, or an analogue thereof, thereby making the nucleotide analogue.

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 NaHCO 3 .

9. The method of claim 4 , wherein the suitable buffered solvent is DMF buffered with NaHCO 3 —Na 2 CO 3 .

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.

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

 wherein F comprises a fluorophore;
(b) removing unbound nucleotide analogues;
(c) determining the identity of the bound nucleotide analogue; and
(d) following step (c), except with respect to the final DNA residue to be sequenced, chemically cleaving from the bound nucleotide analogue the fluorophore and the allyl moiety bound to the 3′-oxygen atom of the deoxyribose,
thereby determining the sequence of the DNA.

13. The method of claim 12 , wherein chemically cleaving the fluorophore and the allyl moiety bound to the 3′-oxygen atom is performed using Na 2 PdCl 4 .

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

wherein F comprises a fluorophore;
(b) reagents suitable for use in DNA polymerization; and
(c) instructions for use.

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

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.

Assignees

• Univ Columbia

Inventors

• Ju Jingyue
• Bi Lanrong
• Kim Dae Hyun
• Meng Qinglin

Classifications

• C07H21/04 Primary

  with deoxyribosyl as saccharide radical · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C09B57/14

  Benzoxanthene dyes; Benzothioxanthene dyes · CPC title