申请人:SHANGHAI EPICAN GENETECH CO. LTD
公开号:US11162139B2
公开(公告)日:2021-11-02
The present invention provides a method for genomic profiling of DNA 5-methylcytosine and 5-hydroxymethylcytosine, comprising the following steps: (1) DNA purification and fragmentation pretreatment: the target DNA is extracted and then broken 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, (4) solid-phase enrichment of the labeled DNA fragments having cytosine with 5-position modification; (5) the PCR 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. The present invention greatly enhances the selectivity and efficiency of binding of the solid-phase surface with the DNA modified base.
本发明提供了一种DNA 5-甲基胞嘧啶和5-羟甲基胞嘧啶基因组谱分析方法,包括以下步骤:(1) DNA 纯化和破碎预处理:提取目标 DNA,然后将其破碎为平均 50 个核苷酸至 10,000 个核苷酸的长度;(2) 修复微量 DNA 并将其与适配体连接:(3)共价标记 5-甲基胞嘧啶和 5-羟甲基胞嘧啶;(4)固相富集具有 5 位修饰胞嘧啶的标记 DNA 片段;(5) 对固相富集的 DNA 片段进行 PCR 扩增,得到 PCR 产物并纯化,得到用于第二代测序的文库,将测序读数映射到基因组后,可生成 5 位修饰胞嘧啶在 DNA 样品中的分布图。本发明大大提高了固相表面与 DNA 修饰碱基结合的选择性和效率。