DIRECTED ENRICHMENT OF GENOMIC DNA FOR HIGH-THROUGHPUT SEQUENCING

• US 20090280540A1
• Filed: 07/16/2009
• Published: 11/12/2009
• Est. Priority Date: 03/23/2006
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: AV Advanced Genetic Analyisi Corporation
• Inventors: Smith, Douglas R., McKernan, Kevin J., Blanchard, Alan
• Granted Patent: US 7,993,842 B2
• Time in Patent Office: Days
• US Class Current: 435/91.200
• 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 method for amplifying a target polynucleotide, comprising:

a) providing a plurality of oligonucleotide primer pairs, wherein each oligonucleotide primer pair is located at a discrete position on a microarray, and wherein each oligonucleotide primer in each oligonucleotide primer pair comprises at least one cleavable linkage;

b) hybridizing a target polynucleotide to at least one primer in at least one oligonucleotide primer pair on the microarray, thereby producing a microarray comprising at least one primer pair comprising at least one primer hybridized to a target polynucleotide;

c) embedding the microarray of step b) into a capturing support;

d) separating the at least one primer oligonucleotide pair comprising at least one primer hybridized to a target polynucleotide from the microarray by cleaving the at least one cleavable linkage in each primer, thereby releasing the at least one oligonucleotide primer pair comprising at least one primer hybridized to a target polynucleotide from the microarray, wherein the at least one oligonucleotide primer pair comprising at least one primer hybridized to a target polynucleotide is captured in the capturing support; and

e) amplifying the target polynucleotide under conditions in which the at least one oligonucleotide primer pair comprising at least one primer hybridized to the target polynucleotide amplifies the target polynucleotide by polymerase chain reaction, whereby an amplified target polynucleotide is produced;

thereby amplifying a target polynucleotide.

2. The method of claim 1, wherein each oligonucleotide primer further comprises at the 5′

end of the oligonucleotide primer a restriction endonuclease recognition site for a distally cleaving restriction endonuclease.

3. The method of claim 2, wherein the distally cleaving restriction endonuclease is a Type IIs or a Type III restriction endonuclease.

4. The method of claim 2, further comprising cleaving the amplified target polynucleotide with a restriction endonuclease specific for the restriction endonuclease recognition site, thereby producing a fragment of the amplified target polynucleotide, wherein at least a portion of the oligonucleotide primer sequence is removed.

5. The method of claim 4, further comprising joining a pair of sequencing adapters to the fragment of the amplified target polynucleotide, wherein one sequencing adapter is joined to each end of the fragment, and wherein each sequencing adapter comprises a primer binding site, thereby producing an adapter-modified target polynucleotide.

6. The method of claim 5, further comprising sequencing the adapter-modified target polynucleotide.

7. The method of claim 5, wherein the pair of sequencing adapters is a pair of asymmetrical adapters, and wherein said pair of asymmetrical adapters comprise:

a) a first oligonucleotide adapter selected from the group consisting of;

(i) an asymmetrical tail adapter comprising a first ligatable end, and a second end comprising a single-stranded 3′

overhang of at least about 8 nucleotides;

(ii) an asymmetrical Y adapter comprising a first ligatable end, and a second unpaired end comprising two non-complementary strands, wherein the length of the non-complementary strands are at least about 8 nucleotides; and

(iii) an asymmetrical bubble adapter comprising an unpaired region of at least about 8 nucleotides flanked on each side by a paired region;

and

b) a second oligonucleotide adapter selected from the group consisting of;

(i) an asymmetrical tail adapter comprising a first ligatable end, and a second end comprising a single-stranded 5′

overhang of at least about 8 nucleotides, wherein the 3′

end of the strand that does not comprise the 5′

overhang comprises at least one blocking group;

(ii) an asymmetrical Y adapter comprising a first ligatable end, and a second unpaired end comprising two non-complementary strands, wherein the length of the non-complementary strands are at least about 8 nucleotides; and

(iii) an asymmetrical bubble adapter comprising an unpaired region of at least about 8 nucleotides flanked on each side by a paired region;

wherein the nucleic acid sequence of the first and second asymmetrical adapters are not identical.

8. The method of claim 1, further comprising joining a universal adapter to each end of the amplified target polynucleotide, wherein each universal adapter comprises a primer binding site, and the 3′

end of each universal adapter comprises a restriction endonuclease recognition site for a distally cleaving restriction endonuclease, thereby producing a first adapter-modified target polynucleotide.

9. The method of claim 8, further comprising amplifying the first adapter-modified target polynucleotide, thereby producing an amplified adapter-modified target polynucleotide.

10. The method of claim 9, further comprising cleaving the amplified adapter-modified target polynucleotide with a restriction endonuclease specific for the restriction endonuclease recognition site, thereby producing a fragment of the amplified adapter-modified target polynucleotide, wherein at least a portion of the universal adapter sequence is removed.

11. The method of claim 10, further comprising joining a pair of sequencing adapters to the fragment of the amplified adapter-modified target polynucleotide, wherein one sequencing adapter is joined to each end of the fragment, and wherein each sequencing adapter comprises a primer binding site, thereby producing a second adapter-modified target polynucleotide.

12. The method of claim 11, further comprising sequencing the second adapter-modified target polynucleotide.

13. The method of claim 11, wherein the pair of sequencing adapters is a pair of asymmetrical adapters, and wherein said pair of asymmetrical adapters comprise:

a) a first oligonucleotide adapter selected from the group consisting of;

(i) an asymmetrical tail adapter comprising a first ligatable end, and a second end comprising a single-stranded 3′

overhang of at least about 8 nucleotides;

(ii) an asymmetrical Y adapter comprising a first ligatable end, and a second unpaired end comprising two non-complementary strands, wherein the length of the non-complementary strands are at least about 8 nucleotides; and

(iii) an asymmetrical bubble adapter comprising an unpaired region of at least about 8 nucleotides flanked on each side by a paired region;

and

b) a second oligonucleotide adapter selected from the group consisting of;

(i) an asymmetrical tail adapter comprising a first ligatable end, and a second end comprising a single-stranded 5′

overhang of at least about 8 nucleotides, wherein the 3′

end of the strand that does not comprise the 5′

overhang comprises at least one blocking group;

(ii) an asymmetrical Y adapter comprising a first ligatable end, and a second unpaired end comprising two non-complementary strands, wherein the length of the non-complementary strands are at least about 8 nucleotides; and

(iii) an asymmetrical bubble adapter comprising an unpaired region of at least about 8 nucleotides flanked on each side by a paired region;

wherein the nucleic acid sequence of the first and second asymmetrical adapters are not identical.

14. The method of claim 1, wherein each oligonucleotide primer further comprises a restriction endonuclease recognition site for a distally cleaving restriction endonuclease, and wherein at least one oligonucleotide primer in the oligonucleotide primer pair further comprises a universal primer sequence.

15. The method of claim 14, wherein the distally cleaving restriction endonuclease is a Type IIs or a Type III restriction endonuclease.

16. The method of claim 14, wherein the capturing support further comprises a primer that is identical to the universal primer sequence.

17. The method of claim 14, wherein the primer that is identical to the universal primer sequence further comprises a polyacrylamide group.

18. The method of claim 17, further comprising amplifying the target polynucleotide under conditions in which the primer that is identical to the universal primer sequence amplifies the target polynucleotide by polymerase chain reaction, thereby producing an amplified target polynucleotide.

19. The method of claim 14, further comprising cleaving the amplified target polynucleotide with a restriction endonuclease specific for the restriction endonuclease recognition site, thereby producing a fragment of the amplified target polynucleotide, wherein at least a portion of the oligonucleotide primer sequences are removed.

20. The method of claim 19, further comprising joining a pair of sequencing adapters to the fragment of the amplified target polynucleotide, wherein one sequencing adapter is joined to each end of the fragment, and wherein each sequencing adapter comprises a primer binding site, thereby producing an adapter-modified target polynucleotide.

21. The method of claim 20, further comprising sequencing the adapter-modified target polynucleotide.

22. The method of claim 20, wherein the pair of sequencing adapters is a pair of asymmetrical adapters, and wherein said pair of asymmetrical adapters comprise:

a) a first oligonucleotide adapter selected from the group consisting of;

(i) an asymmetrical tail adapter comprising a first ligatable end, and a second end comprising a single-stranded 3′

overhang of at least about 8 nucleotides;

(ii) an asymmetrical Y adapter comprising a first ligatable end, and a second unpaired end comprising two non-complementary strands, wherein the length of the non-complementary strands are at least about 8 nucleotides; and

(iii) an asymmetrical bubble adapter comprising an unpaired region of at least about 8 nucleotides flanked on each side by a paired region; and

b) a second oligonucleotide adapter selected from the group consisting of;

(i) an asymmetrical tail adapter comprising a first ligatable end, and a second end comprising a single-stranded 5′

overhang of at least about 8 nucleotides, wherein the 3′

end of the strand that does not comprise the 5′

overhang comprises at least one blocking group;

(ii) an asymmetrical Y adapter comprising a first ligatable end, and a second unpaired end comprising two non-complementary strands, wherein the length of the non-complementary strands are at least about 8 nucleotides; and

(iii) an asymmetrical bubble adapter comprising an unpaired region of at least about 8 nucleotides flanked on each side by a paired region;

wherein the nucleic acid sequence of the first and second asymmetrical adapters are not identical.

23. The method of claim 1, further comprising joining to the ends of the amplified target polynucleotide a pair of asymmetrical adapters, wherein said pair of asymmetrical adapters comprise:

a) a first oligonucleotide adapter selected from the group consisting of;

(i) an asymmetrical tail adapter comprising a first ligatable end, and a second end comprising a single-stranded 3′

overhang of at least about 8 nucleotides;

(ii) an asymmetrical Y adapter comprising a first ligatable end, and a second unpaired end comprising two non-complementary strands, wherein the length of the non-complementary strands are at least about 8 nucleotides; and

(iii) an asymmetrical bubble adapter comprising an unpaired region of at least about 8 nucleotides flanked on each side by a paired region;

and

b) a second oligonucleotide adapter selected from the group consisting of;

(i) an asymmetrical tail adapter comprising a first ligatable end, and a second end comprising a single-stranded 5′

overhang of at least about 8 nucleotides, wherein the 3′

end of the strand that does not comprise the 5′

overhang comprises at least one blocking group;

(ii) an asymmetrical Y adapter comprising a first ligatable end, and a second unpaired end comprising two non-complementary strands, wherein the length of the non-complementary strands are at least about 8 nucleotides; and

(iii) an asymmetrical bubble adapter comprising an unpaired region of at least about 8 nucleotides flanked on each side by a paired region;

wherein the first and second asymmetrical adapters further comprise a restriction endonuclease recognition site for a distally cleaving restriction endonuclease, and wherein the nucleic acid sequence of the first and second asymmetrical adapters are not identical, thereby producing a first adapter-modified target polynucleotide.

24. The method of claim 23, further comprising amplifying the first adapter-modified target polynucleotide, thereby producing an amplified adapter-modified target polynucleotide.

25. The method of claim 24, further comprising cleaving the amplified adapter-modified target polynucleotide with a restriction endonuclease specific for the restriction endonuclease recognition site, thereby producing a fragment of the amplified adapter-modified target polynucleotide, wherein at least a portion of the asymmetrical adapter sequences are removed.

26. The method of claim 25, further comprising joining a pair of sequencing adapters to the fragment of the amplified adapter-modified target polynucleotide, wherein one sequencing adapter is joined to each end of the fragment, and wherein each sequencing adapter comprises a primer binding site, thereby producing a second adapter-modified target polynucleotide.

27. The method of claim 26, further comprising sequencing the second adapter-modified target polynucleotide.

28. The method of claim 26, wherein the pair of sequencing adapters is a pair of asymmetrical adapters, and wherein said pair of asymmetrical adapters comprise:

a) a first oligonucleotide adapter selected from the group consisting of;

(i) an asymmetrical tail adapter comprising a first ligatable end, and a second end comprising a single-stranded 3′

overhang of at least about 8 nucleotides;

(ii) an asymmetrical Y adapter comprising a first ligatable end, and a second unpaired end comprising two non-complementary strands, wherein the length of the non-complementary strands are at least about 8 nucleotides; and

(iii) an asymmetrical bubble adapter comprising an unpaired region of at least about 8 nucleotides flanked on each side by a paired region;

and

b) a second oligonucleotide adapter selected from the group consisting of;

(i) an asymmetrical tail adapter comprising a first ligatable end, and a second end comprising a single-stranded 5′

overhang of at least about 8 nucleotides, wherein the 3′

end of the strand that does not comprise the 5′

overhang comprises at least one blocking group;

(ii) an asymmetrical Y adapter comprising a first ligatable end, and a second unpaired end comprising two non-complementary strands, wherein the length of the non-complementary strands are at least about 8 nucleotides; and

(iii) an asymmetrical bubble adapter comprising an unpaired region of at least about 8 nucleotides flanked on each side by a paired region;

wherein the nucleic acid sequence of the first and second asymmetrical adapters are not identical.

29. The method of claim 1, wherein the at least one target polynucleotide is a fragment of a genomic DNA.

30. The method of claim 29, wherein the fragment of a genomic DNA comprises at least about 1000 nucleotides.

31. The method of claim 1, wherein the semi-solid support is a polyacrylamide gel.

32. The method of claim 31, wherein the polyacrylamide gel is a reversible polyacrylamide gel.

33. A method for amplifying a target polynucleotide, comprising:

a) providing an oligonucleotide primer located at a discrete position on a microarray, wherein the oligonucleotide primer comprises at least one cleavable linkage;

b) hybridizing a target polynucleotide to the oligonucleotide primer on the microarray, wherein the target polynucleotide comprises a universal adapter ligated to each end of the target polynucleotide, and wherein each universal adapter comprises a universal primer sequence, thereby producing a microarray comprising an oligonucleotide primer hybridized to a target polynucleotide;

c) extending the oligonucleotide primer hybridized to the target polynucleotide, thereby producing a first strand primer extension product located at a discrete location on the microarray;

d) removing the target polynucleotide from the first strand primer extension product located at a discrete location on the microarray;

e) embedding the first strand primer extension product located at a discrete location on the microarray into a capturing support, wherein the capturing support comprises a universal primer;

f) amplifying the target polynucleotide in the capturing support under conditions in which the oligonucleotide primer hybridized to the target polynucleotide and the universal primer amplifies the target polynucleotide by polymerase chain reaction, whereby an amplified target polynucleotide is produced;

thereby amplifying a target polynucleotide.

34. The method of claim 33, further comprising cleaving oligonucleotide primer on the microarray after amplifying the target polynucleotide in the capturing support, thereby releasing the amplified target polynucleotide from the microarray.

35. The method of claim 33, wherein the oligonucleotide primer further comprises a restriction endonuclease recognition site for a distally cleaving restriction endonuclease.

36. The method of claim 35, wherein the distally cleaving restriction endonuclease is a Type IIs or a Type III restriction endonuclease.

37. The method of claim 35, further comprising cleaving the oligonucleotide primer with a restriction endonuclease specific for the restriction endonuclease recognition site after amplifying the target polynucleotide in the capturing support, thereby producing a fragment of the amplified target polynucleotide, wherein at least a portion of the oligonucleotide primer sequence is removed.

38. The method of claim 33, further comprising cleaving oligonucleotide primer on the microarray before amplifying the target polynucleotide in the capturing support, thereby releasing the oligonucleotide primer from the microarray.