Phosphite ester oxidation in nucleic acid array preparation

• US 6,833,450 B1
• Filed: 03/15/2001
• Issued: 12/21/2004
• Est. Priority Date: 03/17/2000
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: McGall, Glenn, Kajisa, Lisa
• Primary Examiner(s): EPPS -SMITH, JANET L
• Application Number:
  US09/810,434
• Time in Patent Office: 1,377 Days
• Field of Search: 436/809, 536/23.1, 536/22.1, 536/25.3, 536/25.32, 536/25.31, 435/91.1
• US Class Current: 536/25.3
• 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
• CPC Class Codes: Y10S 436/809 Multifield plates or multicontainer arrays

Abstract

Methods are provided for preparing nucleic acid arrays on a support. In 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 to about 0.05 M iodine in a mixture comprising water and organic solvent.

Claims

1. A method of oxidizing a phosphite ester linkage in a nucleic acid array to a phosphate linkage, comprising contacting said phosphite ester linkage with a solution of from 0.005M to 0.05 M in a mixture of water and organic solvent to form said phosphate linkage.

2. A method of synthesizing a nucleic acid array on a support, wherein each nucleic acid occupies a separate known region of the support, said synthesizing comprising:

(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 each of said other regions has bound thereto another nucleotide comprising a masked reactive site link to a protecting group, wherein said another nucleotide may be the same or different from that used in step (b);

(d) removing the protecting group from one of the nucleotides bound to one of the regions of the support to provide a region beaming a nucleotide having an unmasked reactive site;

(e) binding an additional nucleotide to the nucleotide with an unmasked reactive site,

(f) repeating step (d) and (c) 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 triester linkage between said nucleotides and said unmasked reactive site and further comprising oxidizing said phosphite triester linkage to a phosphate trisect linkage, with a solution of from 0.005 M to 0.05 M in an aqueous solvent mixture.

3. A method in accordance with claim 2, wherein said synthesizing comprises the sequential steps of:

(a) removing a photoremoveable protecting group from at least a first area of a surface of a substrate, said surface comprising immobilized nucleotides on said surface, said nucleotides capped with a photoremoveable protecting group, without removing a photoremoveable protecting group from at least a second area of said surface;

(b) simultaneously contacting said first area and said second area of said surface with a first nucleotide to couple said first nucleotide to said immobilized nucleotides in said first area, and not in said second area, said first nucleotide capped with said photoremoveable protecting group;

(c) removing a photoremoveable protecting group from at least a part of said first area of said surface and at least a pail of said second area;

(d) simultaneously contacting said first area and said second area of said surface with a second nucleotide to couple said second nucleotide to said immobilized nucleotides in at least a part of said first area and at least a part of said second area;

(e) performing additional removing and nucleotide contacting and coupling steps so that a matrix array of at least 100 nucleic acids having different sequences is formed on said support;

with the proviso that the coupling steps further comprise oxidizing an initially formed phosphite ester linkage to a phosphite ester linkage using from 0.005 M to 0.05 M in an aqueous solvent mixture.

4. A method in accordance with claim 3, wherein said aqueous solvent mixture comprises iodine in an amount of 0.02 M.

5. A method in accordance with claim 3, wherein said nucleotides have the formula:

wherein

B is a member selected from the group consisting of natural or unnatural adenine, natural or unnatural guanine, natural or unnatural thymine, natural or unnatural cytosine, and natural or unnatural uracil;

R is a member selected from the group consisting of hydrogen, hydroxy, protected hydroxy, halogen and alkoxy, and

PG is a photoremoveable protecting group.

7. A method in accordance with claim 5, wherein said array comprises at least 10 different nucleic acids.

8. A method in accordance with claim 5, wherein said array comprises at least 100 different nucleic acids.

9. A method in accordance with claim 5, wherein said array comprises at least 1000 different nucleic acids.

10. A method in accordance with claim 5, wherein said array comprises at least 10,000 different nucleic acids.

11. A method in accordance with claim 5, wherein said array comprises at least 100,000 different nucleic acids.

12. A method in accordance with claim 5, wherein each different nucleic acid is in a region having an area of less than about 1 cm².

13. A method in accordance with Claim 5, wherein each different nucleic acid is in a region having an area of less than about 1 mm².

14. A method in accordance with Claim 5, wherein said solution is 0.02 M iodine in a mixture of water, pyridine and THF.

15. A method in accordance with claim 5, wherein B is selected from the group consisting of adenine, guanine, cytosine and thymine, R is hydrogen, and said solution is 0.02 M iodine in a mixture of water, pyridine and THF.

16. A method in accordance with claim 5, wherein B is selected from the group consisting of adenine, guanine, cytosine and thymine, R is hydrogen, PG is McNPOC and said solution is 0.2 M iodine in a mixture of water, pyridine and THF.

17. A method in accordance with Claim 5, wherein B is selected from the group consisting of adenine, guanine, cytosine and thymine, R is hydrogen, PG is McNPOC, the phosphoramidite group is -P(OCH₂CH₂CN)N(iPr)₂ and said solution is 0.02 M iodine in a mixture of water, pyridine and THF.

18. The method of claim 5, wherein from 0.02 M to 0.05 M iodine is present in the aqueous solvent mixture.

6. A method in accordance with Claim S, wherein B is selected from the group consisting of adenine, guanine, cytosine and thymine and R is hydrogen.