Directed enrichment of genomic DNA for high-throughput sequencing

• US 20070231823A1
• Filed: 03/22/2007
• Published: 10/04/2007
• Est. Priority Date: 03/23/2006
• Status: Abandoned Application

• Current Assignee: AB Advanced Genetic Analysis Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: AB Advanced Genetic Analysis Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Smith, Douglas, McKernan, Kevin, Blanchard, Alan
• Application Number:
  US11/726,719
• Publication Number:
  US 20070231823A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2547/107 Use of permeable barriers, e.g. waxes
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

The present invention provides microarrays of oligonucleotide primer pairs, and in particular, microarrays of primers that comprise at least one cleavable linkage. Also provided are methods to capture oligonucleotide primer pairs from one or more microarrays, and methods to use the captured oligonucleotide primer pairs, such as for amplification of a target polynucleotide sequence. In addition, methods of using a microarray to isolate, purify and/or amplify a target polynucleotide are provided.

Claims

1. A microarray comprising a plurality of oligonucleotide primer pairs, wherein each oligonucleotide primer pair comprises a first oligonucleotide primer and a second oligonucleotide primer, each primer comprising at least one cleavable linkage for releasing the plurality of oligonucleotide primer pairs from the microarray.

2. The microarray of claim 1, wherein at least one oligonucleotide primer in each oligonucleotide primer pair comprises a target-specific nucleotide sequence.

3. The microarray of claim 2, wherein at least one oligonucleotide primer in each oligonucleotide primer pair further comprises an affinity tag.

4. The microarray of claim 3, wherein the affinity tag is located at the 5′

terminus of the primer.

5. The microarray of claim 4, wherein the affinity tag is biotin.

6. The microarray of claim 3, wherein at least one oligonucleotide primer in each oligonucleotide primer pair further comprises a universal nucleic acid sequence.

7. The microarray of claim 6, wherein the universal nucleic acid sequence is an M13 sequence.

8. The microarray of claim 1, wherein the oligonucleotide primer pairs are used for PCR.

9. The microarray of claim 1, wherein each element has an area that is at least about 1, 10 or 16 square microns.

10. The microarray of claim 1, wherein the cleavable linkage is a phosphorothiolate linkage, a disulfide linkage, or a deoxyuridine linkage.

11. The microarray of claim 10, wherein the phosphorothiolate linkage is a 5′

phosphorothiolate linkage.

12. The microarray of claim 1, wherein the microarray comprises at least about 100, 1000, 10,000, 100,000, 250,000 or 500,000 distinct elements, wherein each element comprises an oligonucleotide primer pair.

13. The microarray of claim 1, wherein each oligonucleotide primer pair is an oligonucleotide attached to the microarray surface, wherein the oligonucleotide comprises:

a) two primer nucleic acid sequences;

b) a first cleavable linkage located at or near the microarray surface, whereby cleaving the first cleavable linkage separates each oligonucleotide from the microarray surface; and

c) a second cleavable linkage located between the two primer nucleic acid sequences in each oligonucleotide, whereby cleaving the second cleavable linkage separates the two primer nucleic acid sequences.

14. A method for producing a microarray, wherein a plurality of oligonucleotide primer pairs are synthesized on the microarray, each oligonucleotide primer pair is present at a discrete location on the microarray and comprises a first primer and a second primer, and the first primer and second primer each comprise at least one cleavable linkage, comprising:

a) providing a microarray comprising a plurality of discrete locations, wherein each discrete location comprises a first primer synthesis site and a second primer synthesis site;

b) capping each second primer synthesis site with a blocking group;

c) synthesizing a first primer comprising at least one cleavable linkage at each first primer synthesis site on the microarray, thereby producing a plurality of first primers on the microarray;

d) capping the plurality of first primers, thereby preventing further synthesis of each first primer;

e) uncapping each second primer synthesis site; and

f) synthesizing a second primer comprising at least one cleavable linkage at each second primer synthesis site on the microarray, thereby producing a plurality of second primers on the microarray;

thereby producing a microarray, wherein a plurality of oligonucleotide primer pairs are synthesized on the microarray.

15. A microarray produced by the method of claim 14.

16. A method for capturing a plurality of oligonucleotide primer pairs on a capturing means in a capturing support, wherein each oligonucleotide primer pair is present at a discrete location on the capturing support, comprising:

a) embedding each oligonucleotide primer pair present at a discrete location on a microarray into a capturing support, wherein each oligonucleotide primer comprises at least one first cleavable linkage and the capturing support comprises a capturing means for capturing the oligonucleotide primer pairs;

b) capturing the oligonucleotide primer pairs on the microarray by the capturing means; and

c) separating the oligonucleotide primer pairs from the microarray by cleaving the at least one first cleavable linkage;

thereby capturing a plurality of oligonucleotide primer pairs on a capturing means in a capturing support, wherein each oligonucleotide primer pair is present at a discrete location in the capturing support on a capturing means.

26. The method of claim 16, wherein at least a portion of at least one oligonucleotide primer in each oligonucleotide primer pair further comprises a second cleavable linkage, wherein the second cleavable linkage is different from the first cleavable linkage.

27. The method of claim 26, further comprising cleaving at least a portion of the second cleavable linkage of at least one oligonucleotide primer in each oligonucleotide primer pair, thereby producing a capturing support comprising a plurality of oligonucleotide primer pairs, wherein at least a portion of at least one oligonucleotide primer in each oligonucleotide primer pair is liberated from the capturing means in the capturing support.

28. The method of claim 27, further comprising contacting the capturing support comprising a plurality of oligonucleotide primer pairs with a target nucleic acid sequence, and amplifying said target nucleic acid sequence in an amplification reaction, thereby producing an amplified target nucleic acid sequence.

29. The method of claim 28, further comprising sequencing the amplified target nucleic acid sequence.

30. The method of claim 16, wherein each oligonucleotide primer in each oligonucleotide primer pair further comprises a universal primer binding site.

31. The method of claim 16, further comprising removing the microarray from the capturing support after separating the oligonucleotide primer pairs from the microarray.

32. The method of claim 16, wherein the capturing support is a reversible polyacrylamide gel.

33. The method of claim 16, wherein at least one oligonucleotide primer in each oligonucleotide primer pair further comprises a polyacrylamide group.

17-25. -25. (canceled)

34-35. -35. (canceled)

36. A method for capturing a plurality of oligonucleotide primer pairs on a capturing support, wherein each oligonucleotide primer pair comprises a first primer and a second primer, and wherein each first primer is captured from a first microarray and each second primer is captured from a second microarray, comprising:

a) embedding a first microarray comprising a plurality of first primers, wherein each first primer is located on the first microarray at a discrete location, into a capturing support under conditions in which each of the first primers are captured in the capturing support and the discrete location of each of the first primers is maintained in the capturing support, and wherein each first primer comprises at least one first cleavable linkage located at the microarray surface;

b) separating the first primers from the first microarray by cleaving the at least one first cleavable linkage in the first primers;

c) removing the first microarray from the capturing support;

d) embedding a second microarray comprising a plurality of second primers, wherein each second primer is located on the second microarray at a discrete location, into the capturing support under conditions in which each of the second primers are captured in the capturing support and the discrete location of each of the second primers is maintained in the capturing support, wherein the discrete location of each of the second primers overlaps with the discrete location of each of the first primers, and wherein each second primer comprises at least one second cleavable linkage;

e) separating the second primers from the second microarray by cleaving the at least one second cleavable linkage in each of the second primers;

thereby capturing a plurality of oligonucleotide primer pairs on a capturing support, wherein each oligonucleotide primer pair comprises a first primer and a second primer, and wherein each first primer is captured from a first microarray and each second primer is captured from a second microarray.

37. The method of claim 36, further comprising removing the second microarray from the capturing support after the second primers have been separated from the second microarray.

38. The method of claim 36, further comprising contacting the plurality of oligonucleotide primer pairs on the capturing support with a target nucleic acid sequence, and amplifying said target nucleic acid sequence in an amplification reaction, thereby producing a plurality of amplified target nucleic acid sequences.

39. The method of claim 38, wherein the amplified target nucleic acid sequences are sequenced.

40. The method of claim 38, wherein the target nucleic acid sequence is DNA or RNA.

41. The method of claim 36, wherein each oligonucleotide primer in each oligonucleotide primer pair further comprises a universal primer binding site.

42-97. -97. (canceled)