GENERATION OF UNIFORM FRAGMENTS OF NUCLEIC ACIDS USING PATTERNED SUBSTRATES

• US 20120129704A1
• Filed: 04/02/2010
• Published: 05/24/2012
• Est. Priority Date: 04/03/2009
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Lebl, Michal, Heiner, David L., Gunderson, Kevin
• Granted Patent: US 8,709,717 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/101 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Methods of generating nucleic acid fragments of substantially uniform length from sample nucleic acids comprising linearly stretching the sample nucleic acids over a substrate having a plurality of cleavage regions separated by relatively consistent distances, cleaving the linearly stretched sample nucleic acids at the cleavage regions, and collecting the resulting nucleic acid fragments. The method may further include collecting and concentrating the resultant nucleic acid fragments of substantially uniform length.

Claims

1. A method of generating nucleic acid fragments of substantially uniform length from one or more sample nucleic acids, comprising linearly stretching the one or more nucleic acids over a substrate comprising a plurality of cleavage regions separated by relatively consistent distances, and cleaving the one or more linearly stretched sample nucleic acids at the cleavage regions.

4. The method of claim 1, further comprising linearly stretching the one or more sample nucleic acids over the substrate by molecular combing.

5. The method of claim 4, comprising performing the molecular combing in parallel on a plurality of nucleic acid samples.

6. The method of claim 1, further comprising linearly stretching the one or more nucleic acids over the substrate by surface-tethered nucleic acid stretching.

7. The method of claim 6, further comprising attaching a micron-size bead to the free end of the nucleic acid.

8. The method of claim 6, further comprising attaching a magnetic bead to the nucleic acid and using magnets for the nucleic acid stretching.

9. The method of claim 6, comprising performing the surface-tethered nucleic acid stretching in parallel on a plurality of nucleic acid samples.

10. The method of claim 1, wherein the linear stretching of the sample nucleic acid comprises a nucleic acid optical entrapment technique with an optically trapped particle attached to the sample nucleic acid.

11. The method of claim 10, comprising two optically trapped particles attached to the sample nucleic acid, one to each end.

12. The method of claim 1, wherein the linear stretching of the sample nucleic acid comprises electrophoresis of nucleic acid.

13. The method of claim 1, wherein the cleavage regions comprise a nuclease.

14. The method of claim 1, wherein the cleavage regions comprise at least one chemical hydroxyl radical generator.

15. The method of claim 1, wherein the cleavage regions comprise an electrode.

16. The method of claim 1, wherein said cleavage regions are activatable.

17. The method of claim 1, wherein at least one of said one or more samples has a complexity of at least 1 Gbases.

18. The method of claim 1, further comprising collecting the resultant substantially uniform length nucleic acid fragment product.

19. The method of claim 18, further comprising sequencing the resultant substantially uniform length nucleic acid fragment product.

2. A method of generating nucleic acid fragments of substantially uniform length from one or more sample nucleic acids, comprising contacting one or more sample nucleic acids with an affinity ligand, linearly stretching the one or more sample nucleic acids over a substrate including a modified region comprising a plurality of affinity ligand receptors and a plurality of cleavage regions separated by relatively consistent distances, and cleaving the linearly stretched one or more sample nucleic acids at the cleavage regions.

20. The method of claim 2, further comprising linearly stretching the one or more sample nucleic acids over the substrate by molecular combing.

21. The method of claim 20, comprising performing the molecular combing in parallel on a plurality of nucleic acid samples.

22. The method of claim 2, further comprising linearly stretching the one or more nucleic acids over the substrate by surface-tethered nucleic acid stretching.

23. The method of claim 22, further comprising attaching a micron-size bead to the free end of the nucleic acid.

24. The method of claim 22, further comprising attaching a magnetic bead to the nucleic acid and using magnets for the nucleic acid stretching.

25. The method of claim 22, comprising performing the surface-tethered nucleic acid stretching in parallel on a plurality of nucleic acid samples.

26. The method of claim 2, wherein the linear stretching of the sample nucleic acid comprises a nucleic acid optical entrapment technique with an optically trapped particle attached to the sample nucleic acid.

27. The method of claim 26, comprising two optically trapped particles attached to the sample nucleic acid, one to each end.

28. The method of claim 2, wherein the linear stretching of the sample nucleic acid comprises electrophoresis of nucleic acid.

29. The method of claim 2, wherein the cleavage regions comprise a nuclease.

30. The method of claim 2, wherein the cleavage regions comprise at least one chemical hydroxyl radical generator.

31. The method of claim 2, wherein the cleavage regions comprise an electrode.

32. The method of claim 2, wherein said cleavage regions are activatable.

33. The method of claim 2, wherein at least one of said one or more samples has a complexity of at least 1 Gbases.

34. The method of claim 2, further comprising collecting the resultant substantially uniform length nucleic acid fragment product.

35. The method of claim 34, further comprising sequencing the resultant substantially uniform length nucleic acid fragment product.

3. A method of sequencing nucleic acid, comprising linearly stretching one or more sample nucleic acids over a substrate comprising a plurality of cleavage regions separated by a uniform pitch, cleaving the linearly stretched one or more sample nucleic acids at the cleavage regions, thereby producing a substantially uniform length nucleic acid fragment pool, collecting the substantially uniform length nucleic acid fragment pool;

and sequencing the resultant substantially uniform length nucleic acid fragment pool.

36. The method of claim 3, further comprising linearly stretching the one or more sample nucleic acids over the substrate by molecular combing.

37. The method of claim 36, comprising performing the molecular combing in parallel on a plurality of nucleic acid samples.

38. The method of claim 3, further comprising linearly stretching the one or more nucleic acids over the substrate by surface-tethered nucleic acid stretching.

39. The method of claim 38, further comprising attaching a micron-size bead to the free end of the nucleic acid.

40. The method of claim 38, further comprising attaching a magnetic bead to the nucleic acid and using magnets for the nucleic acid stretching.

41. The method of claim 38, comprising performing the surface-tethered nucleic acid stretching in parallel on a plurality of nucleic acid samples.

42. The method of claim 3, wherein the linear stretching of the sample nucleic acid comprises a nucleic acid optical entrapment technique with an optically trapped particle attached to the sample nucleic acid.

43. The method of claim 42, comprising two optically trapped particles attached to the sample nucleic acid, one to each end.

44. The method of claim 3, wherein the linear stretching of the sample nucleic acid comprises electrophoresis of nucleic acid.

45. The method of claim 3, wherein the cleavage regions comprise a nuclease.

46. The method of claim 3, wherein the cleavage regions comprise at least one chemical hydroxyl radical generator.

47. The method of claim 3, wherein the cleavage regions comprise an electrode.

48. The method of claim 3, wherein said cleavage regions are activatable.

49. The method of claim 3, wherein at least one of said one or more samples has a complexity of at least 1 Gbases.