Genome walking by selective amplification of nick-translate DNA library and amplification from complex mixtures of templates

• US 6,777,187 B2
• Filed: 11/15/2001
• Issued: 08/17/2004
• Est. Priority Date: 05/02/2001
• Status: Active Grant

• Current Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Rubicon Genomics, Inc
• Inventors: Kamberov, Emmanuel, Sleptsova, Irina, Makarov, Vladimir L.
• Primary Examiner(s): Siew, Jeffrey
• Application Number:
  US09/999,018
• Publication Number:
  US 20030064376A1
• Time in Patent Office: 1,006 Days
• Field of Search: 435/6, 435/91.2, 435/199, 536/23.1, 536/24.3, 702/20
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Abstract

Improved methods and reagents for chromosome walking of nucleic acid are discussed herein. A library of amplifiable nick translation molecules is generated, and a chromosome walk is initiated from a known sequence in the nucleic acid by producing at least one nick translate molecule, sequencing part of the nick translate molecule, and producing a second nick translate molecule by initiating the primer extension from the region of the obtained sequence of the prior nick translate molecule.

Claims

1. A method of producing a consecutive overlapping series of nucleic acid sequences from a DNA sample, comprising the steps of:

(a) generating a first amplifiable nick translation product, wherein said nick translation of said first amplifiable nick translation product initiates from a known nucleic acid sequence in the DNA sample;

(b) determining at least a partial sequence from said first nick translation product; and

(c) generating at least a second amplifiable nick translation product, wherein said nick translation of said second amplifiable nick translation product initiates from the partial sequence of said first nick translation product.

2. A method of producing a library of consecutive overlapping series of nucleic acid sequences from a DNA sample comprising DNA molecules having a region comprising a known nucleic acid sequence, the method comprising the steps of:

(a) digesting DNA molecules of the DNA sample with a first sequence-specific endonuclease to generate a plurality of DNA fragments;

(b) generating a first amplifiable nick translation product, wherein said nick translation of said first amplifiable nick translation product initiates from the known nucleic acid sequence;

(c) determining at least a partial sequence from said first nick translation product; and

(d) generating one or more additional amplifiable nick translation products, wherein said nick translation of said one or more amplifiable nick translation products initiates from the partial sequence of a previous nick translation product.

3. The method of claim 2, wherein said method further comprises the step of digesting DNA molecules with at least a second sequence-specific endonuclease, wherein an overlapping nick translation product from the one or more additional nick translation products is generated from a DNA fragment from digestion with the first sequence-specific endonuclease or from digestion with the second sequence-specific endonuclease.

4. A method of producing a library of consecutive overlapping series of nucleic acid sequences, comprising the steps of:

(a) obtaining a DNA sample comprising DNA molecules having a region comprising a known nucleic acid sequence;

(b) partially cleaving the DNA molecules with a sequence-specific endonuclease to generate a plurality of DNA ends;

(c) separating the cleaved DNA molecules;

(d) generating a first amplifiable nick translation product, wherein said nick translation of said first amplifiable nick translation product initiates from a known nucleic acid sequence;

(e) determining at least a partial sequence from said first nick translation product; and

(f) generating one or more amplifiable nick translation products, wherein said nick translation of said one or more amplifiable nick translation products initiates from the partial sequence of a previous nick translation product.

5. The method of claim 4, wherein the separation of the cleaved DNA molecules is according to size.

6. The method of claim 5, wherein the size separation is by gel size fractionation.

7. The method of claim 4, wherein the nick translation products are amplified.

8. The method of claim 7, wherein the amplification of the nick translation product comprises polymerase chain reaction utilizing a first primer specific to a known sequence in the nick translation product and a second primer specific to an adaptor sequence of the nick translation product.

9. The method of claim 7, wherein at least one of the nick translation products is selectively amplified from the plurality of nick translation products.

10. The method of claim 7, wherein the nick translation product is single stranded.

11. The method of claim 4, wherein the partial cleavage of the DNA molecules comprises cleaving for a selected time with a frequently cutting sequence-specific endonuclease, wherein the sequence-specificity of the endonuclease is to three or four nucleotide bases.

12. The method of claim 4, wherein the partial cleavage of the DNA molecules comprises subjecting the DNA molecules to a methylase prior to subjection to a methylation-sensitive sequence-specific endonuclease.

13. The method of claim 9, wherein the selective amplification comprises:

(a) introducing to said plurality of nick translation products a plurality of primers, wherein the primers comprise;

(1) nucleotide base sequence complementary to an adaptor sequence in the nick translation product;

(2) an additional variable 3′

terminal nucleotide; and

(3) a label;

(b) hybridizing the primers to their complementary nucleic acid sequences in the adaptor to form a mixture of primer/nick translate molecule hybrids; and

(c) extending from a primer having the 3′

terminal nucleotide complementary to the nucleotide in the nick translate molecule, wherein the nucleotide is immediately adjacent to the adaptor sequence, wherein the hybridizing and extending steps form a mixture of unextended primer/nick translate molecule hybrids and extended primer/nick translate molecule hybrids.

14. The method of claim 13, wherein the method further comprises:

(a) binding of the mixture by the label to a support;

(b) washing the support-bound mixture to remove the nick translate molecules; and

(c) removing the support-bound extended molecule from the support.

15. The method of claim 13, the primer further comprises two or more variable 3′

terminal nucleotides.

16. The method of claim 9, wherein the method further comprises separating the selectively amplified nick translate products by size.

17. The method of claim 16, wherein the size separation is by gel fractionation.

18. The method of claim 16, wherein the method further comprises a step of subjecting the size-separated nick translate molecules to an additional amplification step.

19. The method of claim 9, wherein the selective amplification step is by suppression PCR.

20. The method of claim 19, wherein the suppression PCR utilizes a primer comprising:

(a) a nucleic acid sequence for a primer specific for an adaptor sequence of the nick translate molecule; and

(b) nucleic acid sequence complementary to a region in a plurality of nick translate molecules, whereby the nucleic acid sequence is 5′

to the sequence for a primer specific for an adaptor sequence of the nick translate molecule.

21. The method of claim 9, wherein the at least one selectively amplified nick translate product is amplified by primer extension/ligation reactions.

22. The method of claim 21, wherein the method further comprises immobilization of the nick translation molecules onto a solid support.

23. The method of claim 22, wherein the solid support is a magnetic bead.

24. The method of claim 21, wherein the primer extension/ligation reactions comprise:

(a) initiating and extending the primer extension reaction to form a primer extended molecule, wherein the reaction uses a first primer which is complementary to sequence in a subset of the plurality of nick translate molecules, wherein the complementary sequence of the nick translate molecule is adjacent to a first adaptor end of the nick translate molecule; and

(b) ligating an oligonucleotide to the 5°

end of the extension product, wherein the oligonucleotide comprises sequence complementary to the first adaptor of the nick translate molecule and also comprises a sequence for binding by a second primer, wherein the second primer binding sequence in the oligonucleotide is 5′

to the first adaptor complementary sequence in the oligonucleotide.

25. The method of claim 24, wherein the method further comprises amplifying the primer extended molecule.

26. The method of claim 25, wherein the method further comprises separating the primer extended molecule from the plurality of nick translate molecule.

27. The method of claim 26, wherein the nick translate molecules were generated in the presence of dU nucleotides, the primer extended molecule contains no dU nucleotides, and wherein the separating step comprises degradation of the plurality of nick translate molecules by dU-glycosylase.

28. The method of claim 25, wherein the amplification step comprises polymerase chain reaction using the second primer and a primer complementary to a second adaptor of the nick translate molecule.

29. The method of claim 21, wherein the ligation/primer extension reactions comprise:

(a) ligating in a head-to-tail orientation a plurality of oligonucleotides to form an oligonucleotide assembly, wherein the oligonucleotides are complementary to nick translate molecule sequence that is adjacent to a first adaptor end of the nick translate molecule and wherein the nick translate molecule sequence is present in a subset of the plurality of nick translate molecules, wherein the nick translation molecule has the first adaptor on one terminal end and a second adaptor on the other terminal end;

(b) initiating and extending the primer extension reaction with the 3′

end of the oligonucleotide assembly; and

(c) ligating an oligonucleotide to the 5′

end of the extension product, wherein the oligonucleotide comprises sequence complementary to the first adaptor of the nick translate molecule and also comprises sequence for binding by a first primer, wherein the first primer binding sequence is 5′

to the first adaptor complementary sequence in the oligonucleotide.

30. The method of claim 29, wherein the method further comprises the steps of:

(a) separating the primer extended molecule from the plurality of nick translate molecules; and

(b) amplifying the primer extended molecule.

31. The method of claim 30, wherein the nick translate molecules were generated in the presence of dU nucleotides, the primer extended molecule contains no dU nucleotides, and wherein the separating step comprises degradation of the plurality of nick translate molecules by dU-glycosylase.

32. The method of claim 30, wherein the amplification step comprises polymerase chain reaction using the first primer and a second primer complementary to the second adaptor of the nick translate molecule.

33. The method of claim 21, wherein the primer extension/ligation reaction comprises:

(a) initiating and extending the primer extension reaction with a first primer which is complementary to sequence in a subset of the plurality of nick translate molecules, wherein the nick translate molecule sequence is adjacent to a first adaptor end of the nick translate molecule; and

(b) ligating an oligonucleotide to the 5′

end of the extension product, wherein the oligonucleotide comprises;

(1) sequence complementary to the first adaptor of the nick translate molecule;

(2) sequence for binding by a second primer, wherein the second primer binding sequence is 5′

to the sequence in (1); and

(3) a label at the 5′

end.

34. The method of claim 33, wherein the method further comprises the steps of:

(a) separating the primer extended molecule from the plurality of nick translate molecules by the label of the oligonucleotide; and

(b) amplifying the primer extended molecule.

35. The method of claim 33, wherein the label is biotin.

36. The method of claim 35, wherein the separation further comprises streptavidin-coated magnetic beads.

37. The method of claim 34, wherein the amplification step comprises polymerase chain reaction using the second primer and a third primer complementary to a second adaptor of the nick translate molecule.

38. A method of sequencing nucleic acid, comprising the steps of:

(a) obtaining a DNA sample comprising DNA molecules having a region comprising a known nucleic acid sequence;

(b) partially cleaving the DNA molecules with a sequence-specific endonuclease to generate a plurality of DNA ends;

(c) separating the cleaved DNA molecules;

(d) generating a first amplifiable nick translation product, wherein the first amplifiable nick translation product comprises an adaptor at each end, wherein one adaptor at one end is defined as a first adaptor having a first adaptor sequence and wherein one adaptor at the other end is defined as a second adaptor having a second adaptor sequence, wherein the nick translation of said first amplifiable nick translation product initiates from a known nucleic acid sequence;

(e) determining at least a partial sequence from said first nick translation product;

(f) generating one or more additional amplifiable nick translation products, wherein said nick translation of said one or more additional amplifiable nick translation products initiates from the partial sequence of a previous nick translation product; and

(g) sequencing the nick translation products, wherein the amplified nick translation product is not subjected to cloning prior to the sequencing reaction.

39. The method of claim 38, wherein the DNA sample is a genome.

40. The method of claim 38, wherein there is a limited amount of DNA sample.

41. The method of claim 38, wherein the amplification is by polymerase chain reaction, and one of the primers for the polymerase chain reaction is used as a primer for the sequencing reaction.

42. The method of claim 38, wherein at least a portion of the first adaptor sequence, the second adaptor sequence, or of both first and second adaptor sequences is removed from the amplified nick translation molecule.

43. The method of claim 42, wherein the removal step comprises subjecting the amplified nick translation molecule to a 5′

exonuclease.

44. The method of claim 42, wherein a region of the first adaptor sequence, second adaptor sequence or of both first and second adaptor sequences of the nick translate molecule comprises a dU nucleotide and the removal comprises degradation by dU-glycosylase.

45. The method of claim 42, wherein a region of the first adaptor sequence, second adaptor sequence or of both first and second adaptor sequences comprises a ribonucleotide and the removal comprises degradation by alkaline hydrolysis.

46. The method of claim 44 or 45, wherein the region of the second adaptor sequence is in a 3′

region of the second adaptor sequence.

47. A method of providing sequence for a gap in a genome sequence, comprising the steps of:

(a) obtaining a DNA sample of the genome comprising DNA molecules having a region comprising a known nucleic acid sequence adjacent to the gap;

(b) digesting the DNA molecules with a plurality of sequence-specific endonucleases to generate a plurality of DNA ends;

(c) generating a first amplifiable nick translation product, wherein said nick translation of said first amplifiable nick translation product initiates from the known nucleic acid sequence;

(d) determining at least a partial sequence from said first nick translation product; and

(e) generating one or more additional amplifiable nick translation products, wherein said nick translation of said one or more amplifiable nick translation products initiates from the partial sequence of a previous nick translation product, wherein at least one of the amplifiable nick translation products comprises sequence of the gap.

48. The method of claim 47, wherein the genome is a bacterial genome.

49. The method of claim 47, wherein the genome is a plant genome.

50. The method of claim 47, wherein the genome is an animal genome.

51. The method of claim 50, wherein the animal genome is a human genome.

52. The method of claim 48, wherein the bacteria are unculturable.

53. The method of claim 48, wherein the bacteria is present in a plurality of bacteria.

54. A method of producing a library of consecutive overlapping series of nucleic acid sequences from a DNA sample, comprising the steps of:

(a) obtaining the DNA sample comprising a DNA molecule;

(b) digesting the DNA molecule with a first sequence-specific endonuclease to generate a plurality of DNA fragments, wherein at least one DNA fragment has a region comprising a known nucleic acid sequence;

(c) attaching a first adaptor molecule to ends of the DNA fragments to provide a nick translation initiation site, wherein the first adaptor comprises a label;

(d) subjecting the first adaptor-bound DNA fragment to nick translation comprising DNA polymerization and 5′

-3′

exonuclease activity, wherein the nick translation initiates from the known nucleic acid sequence, to generate a first nick translation product;

(e) isolating the nick translation product by the label;

(f) attaching a second adaptor molecule to the first nick translate product;

(g) determining at least a partial sequence from the first nick translation product; and

(h) generating one or more additional amplifiable nick translation products, wherein said nick translation of said one or more amplifiable nick translation products initiates from the partial sequence of a previous nick translation product.

55. The method of claim 54, wherein the label is biotin and the isolation step is binding to streptavidin-coated magnetic beads.

57. The method of claim 55, wherein the separation of the DNA fragments is by size.

58. The method of claim 57, wherein the size separation is by electrophoresis.

56. A method of producing a library of consecutive overlapping series of nucleic acid sequences, comprising the steps of:

(a) obtaining a DNA sample comprising DNA molecules having a region comprising a known nucleic acid sequence;

(b) partially cleaving the DNA molecules with a sequence-specific endonuclease to generate a plurality of DNA fragments, wherein at least one DNA fragment has a region comprising a known nucleic acid sequence;

(c) separating the cleaved DNA fragments;

(d) attaching a first adaptor molecule to ends of the DNA fragments to provide a nick translation initiation site, wherein the first adaptor comprises a label;

(e) subjecting the first adaptor-bound DNA fragment to nick translation comprising DNA polymerization and 5′

-3′

exonuclease activity, wherein the nick translation initiates from the known nucleic acid sequence, to generate a first nick translation product;

(f) isolating the nick translation product by the label;

(g) attaching a second adaptor molecule to the first nick translate products;

(h) determining at least a partial sequence from said first nick translation product; and

(i) generating one or more additional amplifiable nick translation products, wherein said nick translation of said one or more amplifiable nick translation products initiates from the partial sequence of said first nick translation product.