Phosphite ester oxidation in nucleic acid array preparation

• US 20050119473A1
• Filed: 10/26/2004
• Published: 06/02/2005
• Est. Priority Date: 03/17/2000
• Status: Abandoned Application

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: McGall, Glenn H., Kajisa, Lisa T.
• Application Number:
  US10/974,287
• Publication Number:
  US 20050119473A1
• Time in Patent Office: Days
• US Class Current: 536/25.330
• CPC Class Codes: C07H 1/00 Processes for the preparation of sugar derivatives
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Abstract

Methods are provided for preparing nucleic acid arrays on a support, particularly substantially planar supports. In one group of these methods a plurality of nucleic acids are synthesized on the support and the synthesis steps include oxidizing a phosphite triester nucleic acid linkage to a phosphate triester nucleic acid linkage using a solution about 0.005 M to about 0.05 M iodine in a mixture comprising water and organic solvent. In another group of these methods a plurality of nucleic acids are synthesized on the support and the synthesis steps include oxidizing a phosphite triester nucleic acid linkage to a phosphate triester nucleic acid linkage using a solution comprising iodine and at least 4% by volume of water.

Claims

1. A method of oxidizing a phosphite ester linkage to a phosphate ester linkage in a nucleic acid array formed on a solid support that is substantially planar or comprises substantially planar regions, comprising contacting said phosphite ester linkage with a solution comprising iodine, one or more organic solvents and at least 4% by volume water to form said phosphate ester linkage.

2. The method of claim 1, wherein the solution comprises iodine, one or more organic solvents and from 4% by volume to 50% by volume water.

3. The method of claim 2, wherein the solution comprises iodine, one or more organic solvents and from 4% by volume to 30% by volume water.

4. The method of claim 3, wherein the solution comprises iodine, one or more organic solvents and from 4% by volume to 20% by volume water.

5. A method of synthesizing a nucleic acid array on a support that is substantially planar or comprises substantially planar regions, wherein each nucleic acid occupies a separate known region of the support, comprising the steps of:

a) activating a region of the support;

b) attaching a nucleotide to a first region, said nucleotide having a masked reactive site linked to a protecting group;

c) repeating steps a) and b) on other regions of said support, whereby another nucleotide comprising a masked reactive site linked to a protecting group is bound to each of said other regions, wherein said another nucleotide is the same or different from that used in step b);

e) binding an additional nucleotide to the nucleotide with an unmasked reactive site; and

f) repeating steps d) and e) on regions of the support until a desired plurality of nucleic acids is synthesized, each nucleic acid occupying separate known regions of the support;

wherein said attaching and said binding are each made by covalently forming a phosphite ester linkage between said nucleotides and said unmasked reactive sites and further comprising oxidizing said phosphite ester linkage to a phosphate ester linkage with a solution comprising iodine, one or more organic solvents and at least 4% by volume water.

6. The method of claim 5, further comprising the sequential steps of:

a) removing a photoremoveable protecting group from at least a first region of a surface of a substrate, the surface comprising immobilized nucleotides on the surface, wherein the immobilized nucleotides are capped with a photoremoveable protecting group, thereby activating said first region, without removing a photoremoveable protecting group from at least a second region of said surface;

b) simultaneously contacting the first region and the second region of the surface with a first nucleotide to couple the first nucleotide to the immobilized nucleotide in the first region and not in the second region, wherein the first nucleotide is capped with a photoremoveable protecting group;

c) removing a photoremoveable protecting group from at least a part of the first region of the surface and at least a part of the second region;

d) simultaneously contacting the first region and the second region of the surface with a second nucleotide, to couple the second nucleotide to the immobilized nucleotides in the at least a part of the first region and the at least a part of the second region; and

e) performing additional removing and nucleotide contacting and coupling steps, thereby forming a matrix array of at least 100 nucleic acids having different sequences on the support,

wherein the coupling steps further comprise oxidizing a phosphite ester linkage to a phosphate ester linkage using a solution comprising iodine, one or more organic solvents and at least 4% by volume water.

7. The method of claim 6, wherein the solution comprises iodine, one or more organic solvents and from 4% by volume to 50% by volume water.

8. The method of claim 7, wherein the solution comprises iodine, one or more organic solvents and from 4% by volume to 30% by volume water.

9. The method of claim 8, wherein the solution comprises iodine, one or more organic solvents and from 4% by volume to 20% by volume water.

10. The method of claim 6, wherein the solution comprises 0.005 M to 0.05 M iodine.

11. The method of claim 10, wherein the solution comprises 0.01 M to 0.05 M iodine.

12. The method of claim 6, wherein the first and second nucleotides have the formula:

13. The method of claim 12, wherein B is selected from the group consisting of adenine, guanine, cytosine and thymine and R is hydrogen.

14. The method of claim 6, wherein the array comprises at least 1,000 different nucleic acids.

15. The method of claim 14, wherein the array comprises at least 100,000 different nucleic acids.

16. The method of claim 6, wherein each different nucleic acid is in a known region having an area of less than about 1 cm².

17. The method of claim 16, wherein each different nucleic acid is in a known region having an area of less than about 1 mm².

18. The method of claim 6, wherein the solution comprises 0.01 M to 0.05 M iodine, pyridine, tetrahydrofuran and from 4% by volume to 20% by volume water.