Restriction endonucleases and their applications

• US 8,507,239 B2
• Filed: 02/23/2011
• Issued: 08/13/2013
• Est. Priority Date: 02/23/2010
• Status: Active Grant

• Current Assignee: Thermo Fisher Scientific Baltics, UAB (Thermo Fisher Scientific Incorporated)
• Original Assignee: Fermentas UAB
• Inventors: Lubys, Arvydas, Vitkute, Jolanta, Lubiene, Judita, Janulaitis, Arvydas
• Primary Examiner(s): Leith, Patricia A
• Assistant Examiner(s): Bowers, Erin M
• Application Number:
  US13/032,849
• Publication Number:
  US 20110207139A1
• Time in Patent Office: 902 Days
• Field of Search: 435/196
• US Class Current: 435/196
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Provided is a methylation-specific restriction endonuclease for a DNA duplex substrate, which endonuclease recognizes in a strand of the duplex a 2 to 6 nucleotide recognition sequence comprising a 5-methylcytosine, and cleaves each strand of the duplex at a fixed position outside the recognition sequence.

Claims

1. A methylation-specific restriction endonuclease for a DNA duplex substrate, which endonuclease comprises SEQ ID NO:

1 or a variant thereof at least 90% identical to SEQ ID NO;

1.

2. A methylation-specific restriction endonuclease for a DNA duplex substrate, which endonuclease comprises SEQ ID NO:

1, and recognizes in a strand of the duplex a recognition sequence comprising a 5-methylcytosine, and cleaves each strand of the duplex at a fixed position outside the recognition sequence.

3. The restriction endonuclease according to claim 2, which cleaves the strands of the duplex at positions so as to produce a 5′

overhang of 4 nucleotides.

4. The restriction endonuclease according to claim 2, wherein the recognition sequence is m5CNNG.

5. The restriction endonuclease according to claim 4, which is obtainable from Streptomyces griseoflavus strain RFL11.

6. A method for the site-specific cleavage of double-stranded DNA which contains 5-methylcytosine in one or both strands, which method comprises mixing a sample comprising the double-stranded DNA, a buffer, and a methylation-specific restriction endonuclease according to claim 2 to form a reaction mixture, and incubating the reaction mixture so as to digest the double-stranded DNA completely.

7. The method according to claim 6, wherein the sample further comprises target double-stranded DNA which is free of 5-methylcytosine, which target double-stranded DNA is undigested.

8. A method for determining in a test DNA sample the level of methylation of cytosine at the 5-position, which method comprises:

(i) treating the test DNA sample with a restriction endonuclease according to claim 2 to cleave DNA containing 5-methylcytosine into reaction products;

(ii) incubating the reaction products with a DNA polymerase in the presence of at least one labeled deoxynucleotide or analogue thereof capable of being incorporated into the reaction products by the DNA polymerase; and

(iii) measuring the amount of incorporated label to indicate the level of methylation of cytosine at the 5 position.

9. The method according to claim 8, wherein the amount of incorporated label is measured to indicate the level of methylation of cytosine at the 5 position by comparison with the amount of label incorporated into a control DNA sample which is the same as the test sample (a) without treatment with the restriction endonuclease;

and (b) which was pre-treated with SssI methyltransferase to modify cytosines within CG dinucleotides.

10. The method according to claim 8, wherein the amount of incorporated label is measured to indicate the level of methylation of cytosine at the 5 position by comparison with the amount of label incorporated into a control DNA sample which is the same as the test sample (a) without treatment with the restriction endonuclease, (b) which was digested with a type II restriction endonuclease instead of the methylation-specific restriction endonuclease, or (c) which was digested with a type II restriction endonuclease simultaneously with the methylation-specific restriction endonuclease.

11. The method according to claim 8, wherein the at least one labeled deoxynucleotide or analogue thereof is a DNA synthesis terminator so that only one labeled deoxynucleotide is incorporated into the end of each reaction product.

12. The method according to claim 8, wherein the label comprises a radioactive label, a fluorescent label or a covalently-coupled chemical compound label.

13. A method for genome-wide analysis of individual 5-methylcytosines, which comprises:

(i) treating a DNA sample with a restriction endonuclease according to claim 2 to cleave DNA containing 5-methylcytosine into reaction products,

(ii) incubating the reaction products with a nucleic acid ligase in the presence of a synthetic nucleic acid of known sequence to generate ligated molecules comprising the synthetic nucleic acid and an individual reaction product;

(iii) analyzing the sequence of individual ligated molecules using the nucleotide sequence information from the ligated synthetic nucleic acid; and

(iv) identifying individual 5-methylcytosines which are (a) at the correct distance from the 5′

-end of ligated synthetic nucleic acid, based on the cleavage behaviour of the methylation-specific restriction endonuclease and (b) which occur in the correct sequence context.

14. The method according to claim 13, wherein the reaction products are treated with phosphatase to remove 5′

-phosphates.

15. The method according to claim 13, wherein the nucleic acid ligase is DNA ligase which catalyzes the formation of a phosphodiester bond between juxtaposed 5′

-phosphate and 3′

-hydroxyl termini of nucleic acids.

16. The method according to claim 13, wherein the synthetic nucleic acid is in a single-stranded form.

17. The method according to claim 16, wherein the synthetic nucleic acid has four unspecified bases (5′

-NNNN) at the 5′

end.

18. The method according to claim 13, wherein the synthetic nucleic acid is in a double-stranded linear form.

19. The method according to claim 13, wherein the synthetic nucleic acid is in a double-stranded hairpin form.

20. The method according to claim 13, wherein the synthetic nucleic acid comprises DNA or a mixture of deoxyribonucleotides with other nucleic acids.

21. The method according to claim 13, wherein the synthetic nucleic acid is modified by a covalently coupled fluorescent label or a covalently-coupled chemical compound label.

22. The method according to claim 13, wherein the ligated molecules are amplified.

23. The method according to claim 13, wherein the ligated molecules are treated with bisulfite before sequencing.

24. The method according to claim 23, wherein the ligated molecules are treated with bisulfite before amplification.

25. A method for whole genome analysis of DNA methylation patterns, the method comprising:

(i) treating a DNA sample with at least one restriction endonuclease according to claim 2 to cleave DNA containing 5-methylcytosine into reaction products;

(ii) incubating the reaction products with a nucleic acid ligase in the presence of a synthetic nucleic acid of known sequence to generate ligated molecules;

(iii) amplifying the ligated molecules using the nucleotide sequence information from the ligated synthetic nucleic acid; and

(iv) detecting the amplified products.

26. The method according to claim 25, wherein the reaction products are treated with phosphatase to remove 5′

-phosphates.

27. The method according to claim 25, wherein the nucleic acid ligase is DNA ligase which catalyzes the formation of a phosphodiester bond between juxtaposed 5′

-phosphate and 3′

-hydroxyl termini of nucleic acids.

28. The method according to claim 25, wherein the synthetic nucleic acid is in a single-stranded form.

29. The method according to claim 28, wherein the synthetic nucleic acid has four unspecified bases (5′

-NNNN) at the 5′

end.

30. The method according to claim 25, wherein the synthetic nucleic acid is in a double-stranded linear form.

31. The method according to claim 25, wherein the synthetic nucleic acid is in a double-stranded hairpin form.

32. The method according to claim 25, wherein the synthetic nucleic acid comprises DNA or a mixture of deoxyribonucleotides with other nucleic acids.

33. The method according to claim 25, wherein the ligated molecules are amplified using polymerase chain reaction, isothermal amplification or transcription-mediated amplification.

34. The method according to claim 25, wherein the ligated molecules are treated with bisulfite before amplification.

35. The method according to claim 25, wherein the synthetic nucleic acid is modified by a covalently coupled fluorescent label or a covalently-coupled chemical compound label.

36. The method according to claim 8, wherein the DNA sample is from a single cell.

37. The method according to claim 13, wherein the DNA sample is from a single cell.

38. The method according to claim 25, wherein the DNA sample is from a single cell.

39. The restriction endonuclease according to claim 2, which cleaves the strand comprising the 5-methylcytosine at a position which is 12 nucleotides from the 5-methylcytosine in the 3′

direction.

40. An isolated polypeptide comprising SEQ ID NO:

1 or a variant thereof at least 90% identical to SEQ ID NO;

1.