Nucleic acid array preparation using purified phosphoramidites

• US 20030232361A1
• Filed: 03/27/2003
• Published: 12/18/2003
• Est. Priority Date: 10/26/1993
• Status: Abandoned Application

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Carroll, Robert, Bury, Paul, Barone, Dale, McGall, Glenn, Trulson, Mark
• Application Number:
  US10/402,333
• Publication Number:
  US 20030232361A1
• Time in Patent Office: Days
• US Class Current: 506/16
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00639 the compounds being trapped in or bound to a porous medium
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00711 Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• 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: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 for creating libraries

Abstract

Improved nucleic acid arrays are provided which have been prepared using nucleoside phosphoramidites having reduced levels of interfering phosphoramidite impurities.

Claims

1. A method of preparing a nucleic acid array on a support comprising preparing a nucleic acid array on a support, wherein each nucleic acid occupies a separate known region of the support, said method comprising contacting said support with protected nucleoside phosphoramidite monomers having less than about 1 mole % of a phosphoramidite contaminant, as measured by ¹H NMR spectrometry, selected from the group consisting of (MeO)(NCCH₂CH₂O)PN(iPr)₂, (MeO)P(N(iPr)₂)₂, (MeO)₂PN(iPr)₂, and (NCCH₂CH₂O)₂PN(iPr)₂.

2. A method in accordance with claim 1, said method further comprising:

(a) activating a region of the support;

(b) attaching a nucleotide to said 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 linked to a protecting group, wherein said another nucleotide may be the same or different from that attached 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 bearing a nucleotide having an unmasked reactive site;

(e) binding an additional nucleotide to the nucleotide having 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 phosphoramidite contaminant is present in an amount of less than about 0.5 mole % as measured by ¹H NMR spectrometry.

3. A method in accordance with claim 1, wherein said method comprises the sequential steps of:

(a) generating a pattern of light and dark areas by selectively irradiating at least a first area of a surface of a substrate, said surface comprising immobilized nucleotides on said surface, said nucleotides capped with a photoremovable protecting group, without irradiating at least a second area of said surface, to remove said protecting group from said nucleotides in said first area;

(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 photoremovable protecting group;

(c) generating another pattern of light and dark areas by selectively irradiating with light at least a part of said first area of said surface and at least a part of said second area to remove said protecting group in said at least a part of said first area and said at least a part 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; and

(e) performing additional irradiating 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.

4. A method in accordance with claim 1, wherein said contaminant is present in an amount of less than about 0.2 mole % as measured by ¹H NMR spectrometry.

5. A method in accordance with claim 1, wherein said protected nucleoside phosphoramidite monomers have the formula:

6. A method in accordance with claim 5, wherein B is selected from the group consisting of adenine, guanine, cytosine and thymine and R is hydrogen.

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

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

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

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

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

12. A method in accordance with claim 3, wherein each nucleic acid having a different sequence is in a region having an area of less than about 1 cm².

13. A method in accordance with claim 3, wherein each nucleic acid having a different sequence is in a region having an area of less than about 1 mm².

14. A method in accordance with claim 5, wherein said phosphoramidite contaminant is present in an amount of less than 0.2 mole % as measured by ¹H NMR spectrometry.

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 phosphoramidite contaminant is present in an amount of less than 0.2 mole % as measured by ¹H NMR spectrometry.

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 MeNPOC and said phosphoramidite contaminant is present in an amount of less than 0.2 mole % as measured by ¹H NMR spectrometry.

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 MeNPOC, P is —

P(OCH₂CH₂CN)N(iPr)₂ and said phosphoramidite contaminant is present in an amount of less than 0.2 mole % as measured by ¹H NMR spectrometry.

18. A nucleic acid array prepared by the method of claim 1.

19. A nucleic acid array prepared by the method of claim 5.

20. A nucleic acid array prepared by the method of claim 17.