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METHOD FOR GENOMIC PROFILING OF DNA 5-METHYLCYTOSINE AND 5-HYDROXYMETHYLCYTOSINE

  • US 20170253924A1
  • Filed: 04/26/2017
  • Published: 09/07/2017
  • Est. Priority Date: 03/02/2016
  • Status: Active Grant
First Claim
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1. A method for genomic profiling of DNA 5-methylcytosine and 5-hydroxymethylcytosine, characterized in that, it comprises the following steps:

  • (1) DNA purification and fragmentation pretreatment;

    the target DNA is extracted using a mechanical force or digestive enzyme to break it to an average of 50 nucleotides to 10,000 nucleotides in length;

    (2) the repair of trace amount of DNA and the ligation thereof to the adaptor;

    the pre-treated DNA fragments are repaired and ligated with the sequencing adaptor required for the second-generation sequencing;

    (3) covalently labeling 5-methylcytosine and 5-hydroxymethylcytosine;

    cytosine with 5-position modification in the DNA fragment to which the adaptor is ligated is covalently labeled;

    (4) solid-phase enrichment of the labeled DNA fragment having cytosine with 5-position modification;

    the labeled DNA fragment having cytosine with 5-position modification is bound to the solid phase in the binding buffer, and the solid phase surface is repeatedly washed, to remove the unbound DNA fragment;

    (5) the PCR amplification primers corresponding to the adaptor are used to carry out an amplification of the solid-phase enriched DNA fragments, the PCR product is obtained and purified to obtain a library for the second-generation sequencing, after mapping the sequencing reads to the genome, the distribution map of the cytosine with 5-position modification in the DNA sample could be generated.

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