Parallel preparation of high fidelity probes in an array format

• US 7,547,775 B2
• Filed: 01/03/2006
• Issued: 06/16/2009
• Est. Priority Date: 12/31/2004
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: McGall, Glenn H., Kuimelis, Robert G.
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US11/324,834
• Publication Number:
  US 20060147971A1
• Time in Patent Office: 1,260 Days
• Field of Search: 435/6, 536/23.1, 536/24.3, 536/25.3, 536/25.4, 536/26.6
• US Class Current: 536/25.3
• 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 2219/00427 using masks
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00675 In-situ synthesis on the substrate
• 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: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/14 Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
• CPC Class Codes: C40B 80/00 Linkers or spacers specially adapted for combinatorial chemistry or libraries, e.g. traceless linkers or safety-catch linkers

Abstract

The present invention provides massively parallel oligonucleotide synthesis and purification for applications that utilize large collections of defined high-fidelity oligonucleotides (e.g., from about 10¹ to about 10⁵ different sequences, generally between 25-160 bases in length).

Claims

1. A method of fabricating a plurality of oligonucleotides having free 3′

-hydroxyl groups from a high density oligonucleotide array, said method comprising the steps of

a) providing a solid substrate comprising a plurality of ribonucleotides attached thereto, one said ribonucleotide shown below

2. A method according to claim 1 wherein PG₂ is acetate.

3. A method according to claim 1 wherein PG₃ or PG₄ is a photolabile protecting group.

4. A method according to claim 3 wherein said photolabile protecting group is selected from the group consisting of NNPOC and MBPMOC wherein said photolabile protecting group is attached to the 5′

-hydroxyl group of said 2′

-deoxyribonucleotide as depicted by

5. A method according to claim 1 wherein the 2′

-deoxyribonucleotide has a phosphoramidite reactive group as shown by

6. A method according to any of claims 1-5 wherein B of the 2′

-deoxyribonucleotide is selected from the group consisting of G, A, T, and C.

7. A method according to claim 1 wherein PG₃ or PG₄ is an acid labile protecting group.

8. A method according to claim 7 wherein PG₃ or PG₄ is dimethoxytrityl (DMT).

9. A method according to claim 8 wherein the DMT group is removed in selected areas by exposure to acid generated by a photoacid generator in the presence of electro magnetic radiation of an appropriate wavelength in the presence of an acid scavenger.

10. A method according to claim 9 wherein said acid scavenger is selected from the group consisting of organic bases and polymeric bases.

11. A method according to claim 10 wherein said acid scavenger is a polymeric base.

12. A method according to claim 1 wherein said plurality of oligonucleotides comprises between 10¹ to 10⁵ sets of different sequences, each distinct set of sequences comprising a set.

13. A method according to claim 1 wherein said oligonucleotides are between about 80 to about 160 nucleotides.

14. A method according to claim 1 wherein said density is between 200-2000 pmol/cm².

15. A method according to claim 1 wherein said transesterification is accomplished by raising the pH of the solution to between 9 and 12.

16. A method according to claim 1 wherein said tranesterification is initiated by the addition of metal ions.

17. A method according to claim 1 wherein PG₂ is selected from the group consisting of FPMP, CEE, TBDMS and TOM.

18. A method according to claim 1 wherein said oligonucleotides are probes.

19. A method according to claim 1 wherein said oligonucleotides are primers.

20. A method according to claim 1 wherein between steps d and e unreacted 5′

-hydroxyls are capped.

21. A method according to claim 1 wherein said desired array has between 10¹ to 10⁵ oligonucleotides of different sequences and between about 80-160 bases in length.

22. A method according to claim 1 wherein said release oligonucleotides have authentic 3′

-hydroxy termini upon exposure to a distinct set of conditions or are further processed to have free 3′

hydroxyl termini.

23. A method according to claim 1, further comprising the use of the photoprotective groups NPPOC or NNPOC to provide suitable primer purity and quantity.

24. A method according to claim 1 wherein DMT-based photoresist groups are used to provide fidelity primers.

25. A method according to claim 1 wherein ink-jet based in situ oligonucleotide synthesis is used to provide an oligonucleotide probe.

26. A method according to claim 1 wherein synthesis is initiated with a reverse-orientation RNA monomer that contains an orthogonal 2′

-OH protecting group.

27. A method according to claim 7 wherein following conventional 3′

→

5′

probe synthesis, the 2′

-OH protecting group is removed to allow base-induced intramolecular transesterification.

28. A method according to claim 8 wherein the rate of transesterification is enchanced by raising the pH of the aqueous solution to pH 9-12 or by the addition of particular metal ions.