Method for analyzing dna methylation using next generation sequencer and method for concentrating specific dna fragments

A method for obtaining a DNA fragment group, comprising:

• (1) digesting a DNA to be analyzed with a methylation-sensitive restriction enzyme whose recognition site includes methylated cytosine or cytosine to be methylated and which generates a cohesive end;
• (2) ligating labeled adaptors which do not generate a recognition site of the methylation-sensitive restriction enzyme, to both ends of the DNA fragments obtained in step (1);
• (3) digesting the labeled DNA constructs obtained in step (2) with a methylation-insensitive restriction enzyme which recognizes the same recognition site as the methylation-sensitive restriction enzyme and which generates a cohesive end;
• and
• (4) removing only the labeled DNA fragments from the mixture of DNA fragments obtained in step (3) using a binding partner specific to the label, to obtain a DNA fragment group consisting of DNA fragments in which methylated cytosines are included in both cohesive ends at both ends.

A method for obtaining a DNA fragment group, comprising:

• (1) digesting a DNA to be analyzed with a methylation-sensitive restriction enzyme whose recognition site includes methylated cytosine or cytosine to be methylated and which generates a cohesive end;
• (2) converting both ends of the DNA fragments obtained in step (1) into blunt ends in the presence of labeled deoxynucleoside triphosphate;
• (3) digesting the labeled DNA fragments obtained in step (2) with a methylation-insensitive restriction enzyme which recognizes the same recognition site as the methylation-sensitive restriction enzyme and which generates a cohesive end;
• and
• (4) removing only the labeled DNA fragments from the mixture of DNA fragments obtained in step (3) using a binding partner specific to the label, to obtain a DNA fragment group consisting of DNA fragments in which methylated cytosines are included in both cohesive ends at both ends.

A method for obtaining a DNA fragment group, comprising:

• (1) digesting a DNA to be analyzed with a methylation-sensitive restriction enzyme whose recognition site includes methylated cytosine or cytosine to be methylated and which generates a cohesive end;
• (2) ligating stem-loop adaptors which do not generate a recognition site of the methylation-sensitive restriction enzyme, to both ends of the DNA fragments obtained in step (1);
• (3) digesting the DNA constructs obtained in step (2) with a methylation-insensitive restriction enzyme which recognizes the same recognition site as the methylation-sensitive restriction enzyme and which generates a cohesive end;
• (4) ligating nuclease-resistant labeled adaptors whose 5′-end shows nuclease resistance and which generate the recognition site of the methylation-insensitive restriction enzyme, to each cohesive end of the DNA fragments obtained in step (3);
• and
• (5) treating the DNA constructs obtained in step (4) with a single-strand-specific endonuclease followed by a combination of double-strand-specific and single-strand-specific endonucleases, to completely digest only DNA fragments to which the stem-loop adaptors are ligated, and to obtain a DNA fragment group consisting of DNA fragments to which the nuclease-resistant labeled adaptors are ligated at both ends thereof.

A method for a DNA fragment group, comprising:

• (1) digesting a DNA to be analyzed with a methylation-sensitive restriction enzyme whose recognition site includes methylated cytosine or cytosine to be methylated and which generates a cohesive end;
• (2) ligating nuclease-resistant labeled adaptors whose 5′-end shows nuclease resistance and which have a restriction enzyme recognition site consisting of 8 nucleotides or more and do not generate a recognition site of the methylation-sensitive restriction enzyme, to both ends of the DNA fragments obtained in step (1);
• (3) digesting the DNA constructs obtained in step (2) with a methylation-insensitive restriction enzyme which recognizes the same recognition site as the methylation-sensitive restriction enzyme and which generates a cohesive end;
• (4) ligating stem-loop adaptors to both ends of the DNA fragments obtained in step (3);
• (5) treating the DNA constructs obtained in step (4) with a single-strand-specific endonuclease followed by a combination of double-strand-specific and single-strand-specific endonucleases, to completely digest only DNA fragments to which the stem-loop adaptors are ligated, and to obtain a DNA fragment group consisting of DNA fragments to which the nuclease-resistant labeled adaptors are ligated at both ends thereof.