Controlling use of oligonucleotide sequences released from arrays

• US 7,749,701 B2
• Filed: 08/11/2005
• Issued: 07/06/2010
• Est. Priority Date: 08/11/2005
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Booth, Michael G., Leproust, Eric M
• Primary Examiner(s): Forman; BJ
• Assistant Examiner(s): BHAT, NARAYAN KAMESHWAR
• Application Number:
  US11/203,328
• Publication Number:
  US 20070037175A1
• Time in Patent Office: 1,790 Days
• Field of Search: 435/6, 435/287.2, 536/23.1, 536/24.2, 536/25.3
• US Class Current: 435/6.12
• 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 2219/00454 by chemical cleavage from the solid support
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/18 using a particular method of attachment to the solid support

Abstract

A method for controlling the use of oligonucleotide sequences released from arrays, comprises synthesizing a chemical array of oligonucleotides on a substrate under conditions for producing an array of cleavable oligonucleotides that are blocked from enzymatic reactions after cleavage. Methods may also include receiving a chemical array of cleavable oligonucleotides on a substrate, and cleaving the oligonucleotides from the array, wherein the oligonucleotides are blocked from enzymatic reactions after cleavage. Arrays, populations of oligonucleotides and kits are also provided to facilitate the methods.

Claims

1. A method comprising:

synthesizing a chemical array of single-stranded oligonucleotides on a substrate, wherein each of said single-stranded oligonucleotides comprises a cleavable linker comprising a silanol, wherein cleavage at said silanol releases the single-stranded oligonucleotides from the array to produce released single-stranded oligonucleotides, wherein both the 3′ and

5′

ends of all of the released single-stranded oligonucleotides are unsuitable as substrates for enzymatic reactions.

2. The method of claim 1, wherein the single-stranded oligonucleotides are synthesized in situ on the substrate.

3. The method of claim 1, wherein cleavage of the linker produces a blocked group at the cleavage site.

4. The method of claim 1, wherein the substrate comprises a planar support.

5. The method of claim 1, wherein the substrate comprises one or more beads.

6. The method of claim 1, further comprising cleaving at least a portion of the single-stranded oligonucleotides from the array.

7. The method of claim 6, wherein the portion is cleaved by exposing the array to altered pH conditions.

8. The method of claim 7, wherein single-stranded oligonucleotides are cleaved from the substrate by contacting the array with a base.

9. The method of claim 1, wherein 5′

ends of the single-stranded oligonucleotides are exposed on the array and are blocked with blocking groups which inhibit an enzyme from binding to or catalyzing a reaction at the 5′

ends of the single-stranded oligonucleotides.

10. The method of claim 9, wherein the blocking group comprises a phosphate group.

11. The method of claim 1, wherein 3′

ends of the single-stranded oligonucleotides are exposed on the array and are blocked with blocking groups which inhibit an enzyme from binding to or catalyzing a reaction at the 3′

ends of the single-stranded oligonucleotides.

12. The method of claim 1, wherein the enzymatic reactions comprise a reaction of a kinase, phosphatase, a polymerase, a nuclease, a recombinase, a ligase or a combination thereof.

13. The method of claim 1, wherein the single-stranded oligonucleotides do not comprise 2′

OH groups.

14. The method of claim 1, wherein the single-stranded oligonucleotides do not comprise deoxyuridine bases.

15. The method of claim 1, wherein the single-stranded oligonucleotides do not comprise nucleosides that can be cleaved to generate a reactive end for reacting with an enzyme.

16. The method of claim 1, wherein the single-stranded oligonucleotides do not comprise nucleosides that can be cleaved by a restriction enzyme.

17. The method of claim 1, wherein the single-stranded oligonucleotides are cleaved from the array and provided to a remote location.

18. The method of claim 1, wherein the array is sent to a site that is remote from the site of manufacturing of the array and the single-stranded oligonucleotides are cleaved from the array at the remote site.

19. The method of claim 1, wherein the single-stranded oligonucleotides comprise at least about 10 different sequence single-stranded oligonucleotides.

20. The method of claim 1, further comprising cleaving the single-stranded oligonucleotides and sorting the single-stranded oligonucleotides according to characteristics of the single-stranded oligonucleotides.

21. The method of claim 1, wherein the single-stranded oligonucleotides do not include recognition sites for recombinases.

22. The method of claim 1, wherein the cleavage site is located in a constant domain comprising at least one nucleotide or chemical moiety shared in common with all of the single-stranded oligonucleotides on the array.

23. The method of claim 22, wherein the single-stranded oligonucleotides do not comprise constant domains larger than about 5 nucleotides outside of the cleavage site constant domain.

24. The method of claim 1, wherein the cleavage site comprises a bond between a silanol linker and a nucleotide.

25. A method comprising:

synthesizing a chemical array of single-stranded oligonucleotides on a substrate, wherein each of said single-stranded oligonucleotides comprises a cleavage site, wherein cleavage at said cleavage site releases the single-stranded oligonucleotides from the array, wherein the 5′

ends of the single-stranded oligonucleotides are blocked with a blocking group that comprises a 5′

-OMe-nucleoside phosphoramidite, and wherein both the 3′ and

5′

ends of all of the released single-stranded oligonucleotides are unsuitable as substrates for enzymatic reactions.

26. A method comprising receiving a chemical array of cleavable single-stranded oligonucleotides on a substrate, and cleaving the single-stranded oligonucleotides from the array, wherein both the 3′

and 5′

ends of all the cleaved single-stranded oligonucleotides are blocked from enzymatic reactions after cleavage from the array, wherein said cleavable single-stranded oligonucleotides are attached to said substrate via a cleavable linker that comprises a silanol, and the 5′

ends of the cleavable single-stranded oligonucleotides are blocked with a blocking group that comprises a 5′

-OMe-nucleotiside phosphoramidite.