Compositions, Methods and Related Uses for Cleaving Modified DNA

• US 20100167942A1
• Filed: 12/22/2009
• Published: 07/01/2010
• Est. Priority Date: 12/23/2008
• Status: Abandoned Application

• Current Assignee: New England Biolabs Incorporated
• Original Assignee: New England Biolabs Incorporated
• Inventors: Zheng, Yu, Roberts, Richard J.
• Application Number:
  US12/644,666
• Publication Number:
  US 20100167942A1
• Time in Patent Office: Days
• US Class Current: 506/8
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/331 Methylation site specific nuclease

Abstract

Compositions, methods and related uses are provided relating to cleaving modified DNA. For example, a set of DNA fragments obtainable by enzymatic cleavage of a large DNA is described where at least 50% are similarly sized and have a centrally positioned modified nucleotide. In addition, an enzyme preparation is provided that includes one or more enzymes that recognize a modified nucleotide in a DNA and cleave the DNA at a site that is at a non-random distance from the modified nucleotide. The one or more enzymes are further characterized by an N-terminal conserved domain with greater than 90% amino acid sequence homology to WXD(X)₁₀YXGD. The related uses include creating a methylome, methods of purifying DNA fragments containing a modified nucleotide and diagnostic applications.

Claims

1. A set of double-stranded oligonucleotide fragments obtainable by enzymatic cleavage of a large DNA, the large DNA containing one or more modified nucleotides, the set comprising fragments wherein at least 50% are of a similar size and have a centrally positioned modified nucleotide.

2. A set according to claim 1, wherein the one or more of the fragments are isolated from the set.

3. A set of oligonucleotide fragments according to claim 1, wherein the large DNA is at least 100 nucleotides in length.

4. A set of oligonucleotide fragments according to claim 1, wherein the large DNA is a mammalian genomic DNA.

5. A set of oligonucleotide fragments according to claim 1, wherein the large DNA is human genomic DNA.

6. A set of oligonucleotide fragments according to claim 1, wherein the centrally located modified nucleotide is a cytosine.

7. A set of oligonucleotide fragments according to claim 6, wherein the centrally located modified cytosine is proximate to a guanine.

8. A set of oligonucleotide fragments according to claim 7, wherein the modified cytosine is a methylated or hydroxymethylated cytosine.

9. A set of oligonucleotide fragments according to claim 1, wherein the fragments are less than 60 nucleotides in size.

10. A set of oligonucleotide fragments according to claim 9, wherein the fragments have a similar size in the range of 28-36 nucleotides.

11. A set of oligonucleotide fragments according to claim 1, wherein at least one of the modified nucleotides is located within 30 nucleotides from one end of the fragment.

12. An enzyme preparation, comprising:

at least one enzyme that recognizes a modified nucleotide in a DNA and cleaves the DNA at a site that is distant from the modified nucleotide thereby generating a set of fragments as claimed in claim 1, the at least one enzyme further characterized by an N-terminal conserved domain with greater than 90% amino acid sequence homology to WXD(X)₁₀YXGD.

13. An enzyme preparation according to claim 12, wherein the at least one enzyme cleaves the DNA at a non-random distance from the modified nucleotide.

14. An enzyme preparation according to claim 12, wherein the at least one enzyme has an N-terminal conserved domain with greater than 90% sequence homology with WXD(X)₆G(X)₃YXGD(X)_10-15GN(X)₂L X_10-20PX₃F.

15. An enzyme preparation according to claim 12, wherein the at least one enzyme comprises a recognition domain and a cleavage domain encoded by a single open reading frame.

16. An enzyme preparation according to claim 12, wherein the at least one enzyme has a C-terminal conserved domain with greater than 90% amino acid sequence homology to FEX_20-30DX_2-4DX_19-22(Q/E) XK.

17. An enzyme preparation according to claim 12, wherein the at least one enzyme has an amino acid sequence with greater than 90% sequence homology to a protein sequence selected from SEQ ID NOS:

7-22.

18. An enzyme preparation according to claim 12, wherein the at least one enzyme is fused to an affinity tag.

19. An enzyme preparation according to claim 17, wherein the affinity tag is selected from the group consisting of a chitin-binding domain, maltose-binding domain and a His tag.

20. An enzyme preparation according to claim 12, further comprising an activator DNA.

21. An enzyme preparation according to claim 12, wherein the N-terminal domain is capable of being recognized by an antibody.

22. An antibody as defined in claim 21.

24. A method for obtaining a set of oligonucleotide fragments as claimed in claim 1, comprising:

a. enzymatically cleaving a large DNA containing one or more modified nucleotides; and

b. obtaining the set of oligonucleotide fragments.

25. A method according to claim 24, further comprising separating the set of oligonucleotide fragments from uncleaved DNA.

26. A method according to claim 25, further comprising:

sequencing from the separated set of fragments at least one fragment in the set of fragments to determine the location of one or more modified nucleotides contained within the at least one fragment.

27. A method according to claim 24, further comprising:

analyzing some target oligonucleotide fragments for the presence and location of one or more modified nucleotides in the large DNA.

28. A method according to claim 24, further comprising:

sequencing substantially all the fragments in the set of oligonucleotide fragments and mapping the sequences onto a genome sequence map to determine the location of modified cytosines.

29. A method for identifying one or more isolated enzymes in the enzyme preparation according to claim 12, comprising:

a. searching a sequence database using a sequence selected from the group consisting of SEQ ID. NO;

7-22 and variants thereof; and

b. identifying additional sequences having an N-terminal region characterized by a consensus sequence of WXD(X)₆G (X)₃YXGD(X)_10-15GN(X)₂L X_10-20PX₃F.

30. A method according to claim 29, wherein the identified additional sequences have a C-terminal end comprising a catalytic domain with a consensus sequence of FEX_20-30DX_2-4DX_19-22(Q/E)XK.

31. A method for isolating from a mixture, DNA fragments containing one or more modified nucleotides, comprising:

a. adding to the mixture an enzyme preparation according to claim 12, wherein the at least one enzyme has been mutated so as to lack enzyme cleavage activity, wherein the mutant enzyme is immobilized on a solid surface; and

b. separating the DNA fragments bound to the immobilized enzyme from the mixture.

32. A method to determine the location of at least one modified nucleotide in a large DNA, comprising:

a. cleaving a large DNA with an enzyme preparation according to claim 12;

b. obtaining a set of oligonucleotide fragments, each fragment containing at least one modified nucleotide; and

c. determining the location of the at least one modified nucleotide in a sequence map of the large DNA by sequencing one or more oligonucleotides in the set of oligonucleotide cleavage products.

33. A method of identifying a present or future phenotypic property in a cell preparation or tissue sample from a pattern of modified nucleotides;

comprising;

a. cleaving into fragments a large DNA from a cell preparation or tissue sample by means of an enzyme preparation described in claim 12; and

b. comparing a location for modified nucleotides in the fragments with a pattern of modified nucleotides in a control DNA so as to determine a present or future phenotypic property.

34. A method according to claim 33, wherein (a) further comprises:

separating fragments with one or more modified nucleotides from fragments lacking a modified nucleotide by;

(i) contacting the cleavage fragments with a molecule that binds to the fragments containing the one or more modified nucleotides with an immobilized preparation of an affinity-binding protein;

or

(ii) size separation.

35. A method according to claim 34, wherein the affinity-binding protein is derived from an enzyme preparation according to claim 12, wherein the enzyme cleavage activity of the at least one enzyme has been inactivated.

36. A method according to claim 33, where (a) further comprises:

identifying on a methylome or a genome a location for the one or more modified nucleotides in the immobilized cleavage fragments.

38. A method according to claim 37, wherein the affinity-binding molecule is an enzyme preparation according to claim 12, wherein the enzyme cleavage activity has been inactivated.

39. A method according to claim 38, wherein the isolated enzymes in the enzyme preparation are associated with a binding moiety.

40. A kit, comprising:

an enzyme preparation according to claim 12, in a container and instructions for use.

41. A kit according to claim 40, further comprising an activator molecule.

23. An enzyme preparation, comprising:

one or more enzymes that recognize a modified nucleotide in a DNA and cleave the DNA at a site that is at a non-random distance from the modified nucleotide, the one or more enzymes further characterized by an N-terminal conserved domain with greater than 90% amino acid sequence homology with WXD(X)₁₀YXGD.

37. A method for obtaining a purified preparation of fragments containing one or more modified nucleotides, comprising:

a. contacting a mixture of DNA fragments in which one or more of the fragments contain at least one modified nucleotide with an immobilized preparation of an affinity-binding molecule;

b. binding the one or more fragments containing at least one modified nucleotide to the affinity-binding molecule; and

c. obtaining a purified preparation of fragments containing one or more modified nucleotides.