SYNTHESIS OF FOUR COLOR 3'O-ALLYL, MODIFIED PHOTOCLEAVABLE FLUORESCENT NUCLEOTIDES AND RELATED METHODS

• US 20100041029A1
• Filed: 10/31/2006
• Published: 02/18/2010
• 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, Turro, Nicholas J., Meng, Qinglin, Kim, Dae H., Bi, Lanrong, Bai, Xiaopeng
• Granted Patent: US 7,982,029 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 1/04 Introducing polyphosphoric acid radicals
• CPC Class Codes: C07H 19/067 with ribosyl as the saccharide radical
• 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: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• 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 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

This invention provides a process for making 3′-O-allyl-dGTP-PC-Biodopy-FL-510, 3′-O-allyl-dATP-PC-ROX, 3′-O-allyl-dCTP-PC- and 3′-O-allyl-dUTP-PC-R6G, and related methods.

Claims

1. A method for making 3′

O-allyl-dGTP-PC-Bodipy-FL-510 comprising performing the steps set forth in FIG. 7.

2. A method for making 3′

-O-allyl-dATP-PC-ROX comprising performing the steps set forth in FIG. 8.

3. A method for making 3′

-O-allyl-dCTP-PC-Bodipy-650 comprising performing the steps set forth in FIG. 9.

4. A method for making 3′

-O-allyl-dUTP-PC-R6G comprising performing the steps set forth in FIG. 10.

5. 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 (1) the nucleotide analogues consist of an analogue of dGTP, an analogue of dCTP, an analogue of dTTP or dUTP, and an analogue of dATP, (2) each nucleotide analogue comprises (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine or uracil, and analogues thereof, (ii) a deoxyribose, (iii) a fluorophore photocleavably attached to the base, and (iv) an allyl moiety bound to the 3′

-oxygen of the deoxyribose, so that a nucleotide analogue complementary to the residue being sequenced is bound to the DNA by the DNA polymerase, and (3) each of the four analogues has a predetermined fluorescence wavelength which is different than the fluorescence wavelengths of the other three analogues;

(b) removing unbound nucleotide analogues;

(c) determining the identity of the bound nucleotide analogues; and

(d) following step (c), except with respect to the final DNA residue to be sequenced, (i) chemically cleaving from the bound nucleotide analogue the allyl moiety bound to the 3′

-oxygen atom of the deoxyribose and (ii) photocleaving the fluorophore from the bound nucleotide analogue, wherein steps (d) (i) and (d) (ii) can be performed concurrently or in any order, and step (d) (i) is performed using a Pd catalyst at a pH of about 8.8,

thereby determining the sequence of the DNA.

6. The method of claim 5, wherein chemically cleaving the allyl moiety bound to the 3′

-oxygen atom is performed using Na₂PdCl₄.

7. The method of claim 5, wherein the primer is a self-priming moiety.

8. The method of claim 5, wherein the DNA is bound to a solid substrate.

9. The method of claim 8, wherein the DNA is bound to the solid substrate via 1,3-dipolar azide-alkyne cycloaddition chemistry.

10. The method of claim 8, wherein about 1000 or fewer copies of the DNA are bound to the solid substrate.

11. The method of claim 5, wherein the four fluorescent nucleotide analogues are 3′

-O-allyl-dGTP-PC-Bodipy-FL-510, 3′

-O-allyl-dATP-PC-ROX, 3′

-O-allyl-dCTP-PC-Bodipy-650 and 3′

-O-allyl-dUTP-PC-R6G.

12. The method of claim 5, wherein the DNA polymerase is a 9°

N polymerase.

13. A method for removing an allyl moiety from the 3′

-oxygen of a nucleotide analogue'"'"'s deoxyribose moiety comprising the step of contacting the nucleotide analogue with a Pd catalyst at a pH of about 8.8.

14. The method of claim 13, wherein the Pd catalyst is Na₂PdCl₄.