Parallel preparation of high fidelity probes in an array format

• US 8,034,912 B2
• Filed: 08/12/2010
• Issued: 10/11/2011
• 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:
  US12/855,450
• Publication Number:
  US 20100305006A1
• Time in Patent Office: 425 Days
• Field of Search: 536/23.1, 536/24.3, 536/25.3, 536/25.4, 536/26.6, 435/6
• US Class Current: 536/23.1
• 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 for massively parallel oligonucleotide probe synthesis and release of the synthesized oligonucleotides from an array of probes on a solid substrate, the method comprising:

providing a solid substrate;

attaching a plurality of linkers to the substrate, each linker comprising a cleavable moiety, wherein the cleavable moiety is activatable only at a distinct set of conditions and wherein activation of the cleavable moiety disrupts the linker to allow release of the synthesized oligonucleotides and to provide a substrate with a plurality of attached linkers, wherein the linker has the structure shown below

2. The method of claim 1, wherein the transesterification produces released synthesized oligonucleotides with 3′

hydroxyl groups.

3. The method of claim 1, wherein the distinct set of conditions comprises an elevated pH level.

4. The method of claim 3, wherein the elevated pH level is from about 9 to about 12.

5. The method of claim 1, wherein the distinct set of conditions comprises addition of metal ions.

6. The method of claim 3, wherein the distinct set of conditions further comprises addition of metal ions.

7. The method of claim 1, wherein PG₁ is DMT.

8. The method of claim 7, wherein PG₁ is removed for attachment of the first monomer by exposure to an acid generated by a photoacid generator in the presence of electromagnetic radiation of an appropriate wavelength and in the presence of an acid scavenger.

9. The method of claim 1, wherein PG₂ is selected from the group consisting of:

Ac, FPMP, CEE, TBDMS, TOM, or a photolabile protecting group.

10. The method of claim 9, wherein PG₁ is a photolabile protecting group.

11. The method of claim 10, wherein PG₁ is selected from the group consisting of:

NNPOC or MBPMOC.

12. The method of claim 11, wherein PG₂ is a photolabile protecting group, and wherein the photolabile protecting group of PG₂ is active at wavelengths longer than 365 nm.

13. The method of claim 1, wherein PG₁ is a photolabile protecting group.

14. The method of claim 12, wherein PG₁ is selected from the group consisting of:

NNPOC or MBPMOC.

15. A pool of oligonucleotide probes synthesized according to the method of claim 1, wherein the pool of oligonucleotide probes comprises oligonucleotides of different monomer sequences.

16. The pool of oligonucleotide probes of claim 15, wherein the pool comprises oligonucleotides of 100 different sequences.

17. The pool of oligonucleotide probes of claim 16, wherein the pool comprises oligonucleotides of 100,000 different sequences.

18. The pool of oligonucleotides of claim 15, wherein the different monomer sequences of the oligonucleotide probes are designed for use as polymerase chain reaction primers for a plurality of target sequences.