Method for detecting hydroxylmethylation modification in nucleic acid and use thereof

A method for detecting hydroxymethylation modification in nucleic acid, comprising:

• (i) glycosylating the nucleic acid to obtain a glycosylated nucleic acid having glycosylated hydroxymethylated bases derived from hydroxymethylated bases;
• (ii) respectively digesting a control nucleic acid that is unglycosylated and the glycosylated nucleic acid obtained from the step (1) by using a first restriction endonuclease to produce first control nucleic acid fragments and sample nucleic acid fragments;
• digesting the control nucleic acid or the glycosylated nucleic acid by using a second restriction endonuclease to produce second control nucleic acid fragments;
• (ii) respectively ligating the first control nucleic acid fragments, the sample nucleic acid fragments and the second control nucleic acid fragments that are obtained from the step (2) to a biotin-labeled linker to produce a first control ligated product, a sample ligated product and a second control ligated product with each ligated product having the biotin-labeled linker;
• (iv) respectively digesting the first control ligated product, the sample ligated product and the second control ligated product that are obtained from the step (3) with NlaIII restriction endonuclease to produce a first control NlaIII digested product, a sample NlaIII digested product and a second control NlaIII digested product, with each NlaIII digested product having the biotin-labeled linker at one end and a sticky end at the other end;
• (v) respectively ligating the first control NlaIII digested product, the sample NlaIII digested product and the second control NlaIII digested product that are obtained from the step (4) to a second linker having a recognition site of a specific restriction endonuclease;
• then obtaining a first control secondary-ligated product, a sample secondary-ligated product and a second control secondary-ligated product;
• (vi) respectively digesting the first control secondary-ligated product, the sample secondary-ligated product and the second control secondary-ligated product that are obtained from the step (5) with the specific restriction endonuclease to produce a first control final digested product, a sample final digested product and a second control final digested product, with each final digested product having the second linker at one end and a sticky end at the other end;
• (vii) ligating the first control final digested product, the sample final digested product and the second control final digested product that are obtained from the step (6) to a sequencing adaptor, then amplifying the sequencing adaptor-ligated products to produce a first control sequencing library, a sample sequencing library and a second control sequencing library;
• and
• (viii) sequencing all the libraries obtained from the step (7), analyzing and comparing sequence information to obtain information about hydroxymethylation modification in the nucleic acid.