Restriction endonucleases and their applications

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.