Methods for making nucleotide probes for sequencing and synthesis

• US 8,771,950 B2
• Filed: 08/07/2008
• Issued: 07/08/2014
• Est. Priority Date: 02/07/2006
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Church, George M., Zhang, Kun, Chou, Joseph
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US12/187,460
• Publication Number:
  US 20090099041A1
• Time in Patent Office: 2,161 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2535/113 Cycle sequencing
• 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 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification

Abstract

Compositions and methods for making a plurality of probes for analyzing a plurality of nucleic acid samples are provided. Compositions and methods for analyzing a plurality of nucleic acid samples to obtain sequence information in each nucleic acid sample are also provided.

Claims

1. A method of making a plurality of probes for analyzing a plurality of nucleic acid samples comprising the steps of:

providing linear, single stranded DNA encoding a plurality of probes, wherein a probe includes two regions of homology to target genomic DNA at the ends of the probe and two removable PCR primer regions common to all probes;

converting the linear, single stranded DNA to circular DNA;

amplifying the circular DNA;

releasing the plurality of probes from the amplified DNA; and

removing the removable PCR primer regions from the probes.

2. The method of claim 1, wherein the step of releasing is performed by digestion with a restriction endonuclease or a combination of uracil DNA glycosylase and DNA glycosylase-lyase Endonuclease VIII.

3. The method of claim 1, wherein one or more portions of the probe encode a bar code specific for a nucleic acid sample sequence.

4. The method of claim 1, wherein the step of amplifying the circular DNA is performed by rolling circle amplification.

5. The method of claim 1, wherein prior to the step of converting, the linear, single stranded DNA is amplified using Bst polymerase or Phi29 polymerase.

6. The method of claim 1, wherein prior to the step of converting, the linear, single stranded DNA is amplified by PCR.

7. The method of claim 1, wherein prior to the step of converting, the linear, single stranded DNA is purified by size selection.

8. The method of claim 1, wherein the plurality of nucleic acid samples are selected from the group consisting of genomic DNA regions, mRNA and cDNA.

9. The method of claim 1, wherein the linear, single stranded DNA is synthesized on a DNA chip.

10. The method of claim 1, wherein one or more portions of the probe encode a bar code specific for a patient.

11. The method of claim 1, wherein the step of converting the linear, single stranded DNA to circular DNA is performed using a DNA ligase.

12. A method of making a plurality of probes comprising the steps of:

providing linear, single stranded DNA encoding a plurality of probes, wherein a probe includes two regions of homology to target genomic DNA at the ends of the probe and two PCR primer regions common to all probes;

converting the linear, single stranded DNA to circular DNA;

amplifying the circular DNA to form amplified double stranded, circular DNA;

converting the amplified double stranded, circular DNA to single stranded, circular DNA; and

releasing the plurality of probes from the single stranded, circular DNA.

13. The method of claim 12, wherein the step of releasing is performed by digestion with a restriction endonuclease.

14. The method of claim 12, wherein one or more portions of the probe further encode a bar code specific for a nucleic acid sample sequence.

15. The method of claim 12, wherein prior to the step of converting, the linear, single stranded DNA is amplified by PCR.

16. The method of claim 12, wherein prior to the step of converting, the linear, single stranded DNA is purified by size selection.

17. The method of claim 12, wherein the linear, single stranded DNA is obtained from a sample selected from the group consisting of genomic DNA regions, mRNA and cDNA.

18. The method of claim 17, wherein the genomic DNA regions are selected from the group consisting of exons, single nucleotide polymorphisms, mutable regions and highly conserved regions.

19. The method of claim 12, wherein the linear, single stranded DNA is synthesized on a DNA chip.

20. The method of claim 12, wherein the PCR primer regions are removed from the probes by digestion with a restriction endonuclease or a combination of uracil DNA glycosylase and DNA glycosylase-lyase Endonuclease VIII.

21. The method of claim 12, wherein one or more portions of the probe further encode a bar code specific for a patient.

22. The method of claim 12, wherein a probe includes two regions of homology to one or more targets selected from the group consisting of an exon, a single nucleotide polymorphism, a mutable region and a conserved region.

23. The method of claim 12, wherein the step of converting the linear, single stranded DNA to circular DNA is performed using a DNA ligase.

24. A method of making a renewable pool of probes for analyzing a plurality of nucleic acid samples comprising the steps of:

providing a plurality of linear, single stranded DNA probes, wherein a probe includes two regions of homology to target genomic DNA at the ends of the probe and two removable PCR primer regions common to all probes;

converting the linear, single stranded DNA to circular DNA;

amplifying the circular DNA by rolling circle amplification to form linear concatemers;

removing the removable PCR primer regions from the probes;

digesting the linear concatemers to form monomers; and

ligating the monomers to form a plurality of circular molecules complementary to either the plus strand or the minus strand of the circular DNA.

25. The method of claim 24, wherein the plurality of linear, single stranded DNA probes are synthesized on a DNA chip.

26. The method of claim 24, wherein a probe further includes a unique endonuclease recognition site and the linear concatemers are digested with an endonuclease in the presence of a guide oligonucleotide.

27. A method of making a plurality of probes for analyzing a plurality of nucleic acid samples comprising the steps of:

providing linear, single stranded DNA encoding a plurality of probes, wherein a probe includes two regions of homology to target genomic DNA at the ends of the probe and two removable PCR primer regions common to all probes;

converting the linear, single stranded DNA to circular DNA;

amplifying the circular DNA;

releasing the plurality of probes from the amplified DNA, and

wherein the PCR primer regions are removed from the probes by digestion with a restriction endonuclease or a combination of uracil DNA glycosylase and DNA glycosylase-lyase Endonuclease VIII.

28. The method of claim 27, wherein the step of releasing is performed by digestion with a restriction endonuclease or a combination of uracil DNA glycosylase and DNA glycosylase-lyase Endonuclease VIII.

29. The method of claim 27, wherein one or more portions of the probe encode a bar code specific for a nucleic acid sample sequence.

30. The method of claim 27, wherein the step of amplifying the circular DNA is performed by rolling circle amplification.

31. The method of claim 27, wherein prior to the step of converting, the linear, single stranded DNA is amplified using Bst polymerase or Phi29 polymerase.

32. The method of claim 27, wherein prior to the step of converting, the linear, single stranded DNA is amplified by PCR.

33. The method of claim 27, wherein prior to the step of converting, the linear, single stranded DNA is purified by size selection.

34. The method of claim 27, wherein the plurality of nucleic acid samples are selected from the group consisting of genomic DNA regions, mRNA and cDNA.

35. The method of claim 27, wherein the linear, single stranded DNA is synthesized on a DNA chip.

36. The method of claim 27, wherein one or more portions of the probe encode a bar code specific for a patient.

37. The method of claim 27, wherein the step of converting the linear, single stranded DNA to circular DNA is performed using a DNA ligase.

38. A method of making a renewable pool of probes for analyzing a plurality of nucleic acid samples comprising the steps of:

providing a plurality of linear, single stranded DNA probes, wherein a probe includes two regions of homology to target genomic DNA at the ends of the probe and two removable PCR primer regions common to all probes;

converting the linear, single stranded DNA to circular DNA;

amplifying the circular DNA by rolling circle amplification to form linear concatemers;

removing the PCR primer regions from the probes by digestion with a restriction endonuclease or a combination of uracil DNA glycosylase and DNA glycosylase-lyase Endonuclease VIII;

digesting the linear concatemers to form monomers; and

ligating the monomers to form a plurality of circular molecules complementary to either the plus strand or the minus strand of the circular DNA.

39. The method of claim 38, wherein the plurality of linear, single stranded DNA probes are synthesized on a DNA chip.

40. The method of claim 38, wherein a probe further includes a unique endonuclease recognition site and the linear concatemers are digested with an endonuclease in the presence of a guide oligonucleotide.

41. A method of making a plurality of probes for analyzing a plurality of nucleic acid samples comprising the steps of:

providing linear, single stranded DNA encoding a plurality of probes, wherein a probe includes two regions of homology to target genomic DNA at the ends of the probe and two removable PCR primer regions common to all probes, and wherein one or more portions of the probe encode a bar code specific for a patient;

converting the linear, single stranded DNA to circular DNA;

amplifying the circular DNA; and

releasing the plurality of probes from the amplified DNA.

42. The method of claim 41, wherein the step of releasing is performed by digestion with a restriction endonuclease or a combination of uracil DNA glycosylase and DNA glycosylase-lyase Endonuclease VIII.

43. The method of claim 41, wherein one or more portions of the probe encode a bar code specific for a nucleic acid sample sequence.

44. The method of claim 41, wherein the step of amplifying the circular DNA is performed by rolling circle amplification.

45. The method of claim 41, wherein prior to the step of converting, the linear, single stranded DNA is amplified using Bst polymerase or Phi29 polymerase.

46. The method of claim 41, wherein prior to the step of converting, the linear, single stranded DNA is amplified by PCR.

47. The method of claim 41, wherein prior to the step of converting, the linear, single stranded DNA is purified by size selection.

48. The method of claim 41, wherein the plurality of nucleic acid samples are selected from the group consisting of genomic DNA regions, mRNA and cDNA.

49. The method of claim 41, wherein the linear, single stranded DNA is synthesized on a DNA chip.

50. The method of claim 41, wherein the PCR primer regions are removed from the probes by digestion with a restriction endonuclease or a combination of uracil DNA glycosylase and DNA glycosylase-lyase Endonuclease VIII.

51. The method of claim 41, wherein the step of converting the linear, single stranded DNA to circular DNA is performed using a DNA ligase.

52. A method of making a renewable pool of probes for analyzing a plurality of nucleic acid samples comprising the steps of:

providing a plurality of linear, single stranded DNA probes, wherein a probe includes two regions of homology to target genomic DNA at the ends of the probe and two removable PCR primer regions common to all probes and wherein one or more portions of the probe encode a bar code specific for a patient;

converting the linear, single stranded DNA to circular DNA;

amplifying the circular DNA by rolling circle amplification to form linear concatemers;

digesting the linear concatemers to form monomers; and

ligating the monomers to form a plurality of circular molecules complementary to either the plus strand or the minus strand of the circular DNA.

53. The method of claim 52, wherein the plurality of linear, single stranded DNA probes are synthesized on a DNA chip.

54. The method of claim 52, wherein a probe further includes a unique endonuclease recognition site and the linear concatemers are digested with an endonuclease in the presence of a guide oligonucleotide.