Compositions and methods for analysis of nucleic acids

• US 6,762,022 B2
• Filed: 03/06/2001
• Issued: 07/13/2004
• Est. Priority Date: 03/05/1997
• Status: Expired due to Term

• Current Assignee: Board of Regents of the University of Michigan (University of Michigan)
• Original Assignee: Board of Regents of the University of Michigan (University of Michigan)
• Inventors: Makarov, Vladimir L., Langmore, John P.
• Primary Examiner(s): Horlick, Kenneth
• Assistant Examiner(s): Kim, Young J.
• Application Number:
  US09/801,346
• Publication Number:
  US 20020042059A1
• Time in Patent Office: 1,225 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.2, 536/24.3, 530/300
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]

Abstract

Disclosed are a number of methods that can be used in a variety of embodiments, including, creation of a nucleic acid terminated at one or more selected bases, sequence analysis of nucleic acids, mapping of sequence motifs within a nucleic acid, positional mapping of nucleic acid clones, and analysis of telomeric regions. The methods utilize double-stranded templates, and in most aspects involve a strand replacement reaction initiated at one or more random or specific locations created in a nucleic acid molecule, and in certain aspects utilizing an oligonucleotide primer.

Claims

1. A method for preparing a DNA molecule comprising the steps of:

a) obtaining a sample of DNA wherein the sample includes DNA fragments that do not include a 3′

hydroxyl group;

b) conditioning DNA fragments of the sample to provide a 3′

hydroxyl group thereon; and

c) attaching a double stranded oligonucleotide adaptor to only one strand of the conditioned DNA fragment.

2. The method of claim 1, wherein DNA molecules of the DNA sample have been fragmented.

3. The method of claim 2, wherein the DNA molecules have been fragmented by chemical means.

4. The method of claim 3, wherein the DNA molecules have been fragmented through a reaction that includes hydroxyl radicals.

5. The method of claim 4, wherein the DNA molecules have been fragmented through treatment with a Fenton reagent.

6. The method of claim 5, wherein the Fenton reagent comprises a metal ion chelating agent and a divalent metal ion.

7. The method of claim 2, wherein the DNA molecules have been fragmented by enzymatic means.

8. The method of claim 7, wherein the DNA molecules have been fragmented using an endonuclease.

9. The method of claim 8, wherein the endonuclease introduced a blunt end.

10. The method of claim 7, wherein the DNA molecules have been fragmented through the use of a restriction endonuclease.

11. The method of claim 10, wherein the DNA molecules have been fragmented through the use of a restriction endonuclease having a two base recognition sequence.

12. The method of claim 10, wherein the DNA molecules have been fragmented through the use of a restriction endonuclease having a four base recognition sequence.

13. The method of claim 10, wherein the restriction endonuclease has introduced random double strand breaks into DNA molecules.

14. The method of claim 2, wherein the DNA molecules have been fragmented by physical means.

15. The method of claim 14, wherein the DNA molecules have been fragmented by sonication.

16. The method of claim 14, wherein the DNA molecules have been fragmented by nebulization.

17. The method of claim 14, wherein the DNA molecules have been fragmented by hydrodynamic shear.

18. The method of claim 14, wherein the DNA molecules have been fragmented by freezing and thawing.

19. The method of claim 1, wherein DNA fragments that lack a 3′

hydroxyl are conditioned through the use of a 3′

exonuclease.

20. The method of claim 19, wherein the 3′

exonuclease is exonuclease III.

21. The method of claim 1, wherein the DNA fragments that lack a 3′

hydroxyl are conditioned through the use of a DNA polymerase that possesses 3′

to 5′

exonuclease activity.

22. The method of claim 1, wherein the double stranded adaptor is attached to the conditioned DNA by means of a 5′

terminus of the adaptor.

23. The method of claim 22, wherein the double-stranded oligonucleotide adaptor is blocked at at least one of its 3′

termini.

24. The method of claim 23, wherein the double-stranded oligonucleotide adaptor is blocked at both of its 3′

termini.

25. The method of claim 1, wherein the conditioned DNA fragments are amplified.

26. The method of claim 25, wherein DNA fragments are amplified through a PCR reaction.

27. The method of claim 26, wherein the DNA fragments are amplified through a PCR reaction through the use of double-stranded adaptors that have been attached to the conditioned DNA fragments.

28. The method of claim 1, further defined as comprising the steps of:

a) obtaining a sample of DNA wherein the sample includes DNA fragments that do not include a 3′

hydroxyl group, wherein the sample has been subjected to fragmentation;

b) conditioning DNA fragments of the sample that lack 3′

hydroxyl by incorporating a 3′

hydroxyl group thereon;

c) attaching double stranded oligonucleotide adaptors to DNA fragments of the sample; and

d) amplifying DNA fragments of the sample through the use of said double stranded oligonucleotide adaptors.

29. A method for preparing a DNA molecule comprising the steps of:

a) obtaining a sample of DNA wherein the sample includes DNA fragments that do not include a 3′

hydroxyl group, wherein the DNA molecules have been fragmented by chemical means; and

b) conditioning DNA fragments of the sample to provide a 3′

hydroxyl group thereon.

30. The method of claim 29, wherein the DNA molecules have been fragmented through a reaction that includes hydroxyl radicals.

31. The method of claim 30, wherein the DNA molecules have been fragmented through treatment with a Fenton reagent.

32. The method of claim 31, wherein the Fenton reagent comprises a metal ion chelating agent and a divalent metal ion.

33. The method of claim 29, wherein DNA fragments that lack a 3′

hydroxyl are conditioned through the use of a 3′

exonuclease.

34. The method of claim 33, wherein the 3′

exonuclease is exonuclease III.

35. The method of claim 29, wherein the DNA fragments that lack a 3′

hydroxyl are conditioned through the use of a DNA polymerase that possesses 3′

to 5′

exonuclease activity.

36. The method of claim 29, further comprising attaching an oligonucleotide adaptor to the conditioned DNA fragments.

37. The method of claim 36, wherein the oligonucleotide adaptor is a double-stranded oligonucleotide adaptor.

38. The method of claim 37, wherein the double-stranded oligonucleotide adaptor is attached to the conditioned DNA by only one of its two strands.

39. The method of claim 38, wherein the double stranded adaptor is attached to the conditioned DNA by means of a 5′

terminus of the adaptor.

40. The method of claim 39, wherein the double-stranded oligonucleotide adaptor is blocked at at least one of its 3′

termini.

41. The method of claim 40, wherein the double-stranded oligonucleotide adaptor is blocked at both of its 3′

termini.

42. The method of claim 29, wherein the conditioned DNA fragments are amplified.

43. The method of claim 42, wherein DNA fragments are amplified through a PCR reaction.

44. The method of claim 43, wherein the DNA fragments are amplified through a PCR reaction through the use of double-stranded adaptors that have been attached to the conditioned DNA fragments.

45. The method of claim 29, further defined as comprising the steps of:

a) obtaining a sample of DNA wherein the sample includes DNA fragments that do not include a 3′

hydroxyl group, wherein the sample has been subjected to fragmentation;

b) conditioning DNA fragments of the sample that lack a 3′

hydroxyl by incorporating a 3′

hydroxyl group thereon;

c) attaching adaptors to DNA fragments of the sample; and

d) amplifying DNA fragments of the sample through the use of the adaptors.