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1.发明授权Method for detecting hydroxylmethylation modification in nucleic acid and use thereof 有权检测核酸中羟基甲基化修饰的方法及其用途公开(公告)号：US09567633B2
公开(公告)日：2017-02-14
申请号：US14360594
申请日：2012-11-21
发明人： Fei Gao , Junwen Wang , Xiuqing Zhang , Huanming Yang
IPC分类号： C12Q1/68 , C12Q1/54 , C12P19/18 , C12M1/34 , C12N15/74 , G01N31/22 , G01N33/52 , C07H21/04 , C40B40/06
CPC分类号： C12Q1/6874 , C12Q1/683 , C12Q2600/154 , C12Q2521/501 , C12Q2521/531 , C12Q2525/197 , C12Q2537/164 , C12Q2563/131 , C12Q2521/331
摘要： A method for detecting hydroxymethylation modification in nucleic acid comprises: glycosylating the nucleic acid, digesting with MspI, ligating the digested fragments to a biotin-labeled linker at both ends thereof, digesting with NlaIII; capturing the digested fragments using streptavidin magnetic beads to produce fragments having the biotin-labeled linker at one end and a CATG 4-base sticky end at the other end, wherein these fragments reveal modification information of their adjacent CCGG sites; ligating the CATG sticky end to a linker containing a recognition site of MmeI or Ecop15I, digesting with corresponding restriction endonuclease to produce short sequence fragments that can reveal modification information of their adjacent CCGG sites; and performing a tag number comparison to obtain information about methylation and hydroxymethylation modification relative levels. A use of the method is also provided.
摘要翻译： 用于检测核酸中羟基甲基化修饰的方法包括：使核酸糖基化，用MspI消化，将消化的片段两端连接到生物素标记的接头，用NlaIII消化; 使用链霉抗生物素蛋白磁珠捕获消化的片段，以在另一端产生具有生物素标记的接头的片段和另一端的CATG 4-碱基粘性末端，其中这些片段揭示其相邻CCGG位点的修饰信息; 将CATG粘性末端连接到含有MmeI或Ecop15I的识别位点的接头，用相应的限制性内切核酸酶消化以产生可以揭示其相邻CCGG位点的修饰信息的短序列片段; 并进行标签号比较以获得关于甲基化和羟甲基化修饰相关水平的信息。 还提供了该方法的使用。
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2.发明申请公开(公告)号：US20140329697A1
公开(公告)日：2014-11-06
申请号：US14358674
申请日：2012-11-15
发明人： Fei Gao , Junwen Wang , Tong Wang , Hui Jiang , Jinghua Wu , Honglong Wu
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6869 , C12N15/1093 , C12Q1/6806 , C12Q1/6876 , C40B40/06 , C40B40/08 , C40B50/06 , C12Q2523/125 , C12Q2525/117 , C12Q2525/191 , C12Q2535/122
摘要： The present invention provides a method for constructing a high-throughput sequencing library, which comprises: fragmenting genomic DNA; end-repairing the DNA fragments; adding a base A to the 3′ end of the end-repaired DNA fragments; connecting the DNA fragments having cohesive end A with a methylated adapter; carrying out hybrid capture on the connection products by using specific probes to obtain object fragments; treating the object fragments with bisulfite, to convert non-methylated cytosines to uracils; PCR amplifying the converted object fragments; and separating and purifying the amplification products, wherein the amplification products constitute the high-throughput sequencing library. The present invention also provides a method and an apparatus for identifying methylation information in specified genome regions of a sample.
摘要翻译： 本发明提供了构建高通量测序文库的方法，其包括：基因组DNA的片段化; 最终修复DNA片段; 在最终修复的DNA片段的3'端加入碱基A; 将具有粘性末端A的DNA片段与甲基化衔接子连接; 通过使用特定的探针对连接产品进行混合捕获以获得对象片段; 用亚硫酸氢盐处理物体碎片，将非甲基化的胞嘧啶转化成尿嘧啶; PCR扩增转化的对象片段; 并分离和纯化扩增产物，其中扩增产物构成高通量测序文库。 本发明还提供了用于鉴定样品的指定基因组区域中的甲基化信息的方法和装置。
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公开(公告)号：US09920363B2
公开(公告)日：2018-03-20
申请号：US14358674
申请日：2012-11-15
发明人： Fei Gao , Junwen Wang , Tong Wang , Hui Jiang , Jinghua Wu , Honglong Wu
CPC分类号： C12Q1/6869 , C12N15/1093 , C12Q1/6806 , C12Q1/6876 , C40B40/06 , C40B40/08 , C40B50/06 , C12Q2523/125 , C12Q2525/117 , C12Q2525/191 , C12Q2535/122
摘要： The present invention provides a method for constructing a high-throughput sequencing library, which comprises: fragmenting genomic DNA; end-repairing the DNA fragments; adding a base A to the 3′ end of the end-repaired DNA fragments; connecting the DNA fragments having cohesive end A with a methylated adapter; carrying out hybrid capture on the connection products by using specific probes to obtain object fragments; treating the object fragments with bisulfite, to convert non-methylated cytosines to uracils; PCR amplifying the converted object fragments; and separating and purifying the amplification products, wherein the amplification products constitute the high-throughput sequencing library. The present invention also provides a method and an apparatus for identifying methylation information in specified genome regions of a sample.
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4.发明申请METHOD FOR DETECTING HYDROXYLMETHYLATION MODIFICATION IN NUCLEIC ACID AND USE THEREOF 有权用于检测核酸中羟基甲基化修饰的方法及其用途公开(公告)号：US20150031552A1
公开(公告)日：2015-01-29
申请号：US14360594
申请日：2012-11-21
发明人： Fei Gao , Junwen Wang , Xiuqing Zhang , Huanming Yang
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6874 , C12Q1/683 , C12Q2600/154 , C12Q2521/501 , C12Q2521/531 , C12Q2525/197 , C12Q2537/164 , C12Q2563/131 , C12Q2521/331
摘要： A method for detecting hydroxymethylation modification in nucleic acid comprises: glycosylating the nucleic acid, digesting with MspI, ligating the digested fragments to a biotin-labeled linker at both ends thereof, digesting with NlaIII; capturing the digested fragments using streptavidin magnetic beads to produce fragments having the biotin-labeled linker at one end and a CATG 4-base sticky end at the other end, wherein these fragments reveal modification information of their adjacent CCGG sites; ligating the CATG sticky end to a linker containing a recognition site of MmeI or Ecop15I, digesting with corresponding restriction endonuclease to produce short sequence fragments that can reveal modification information of their adjacent CCGG sites; and performing a tag number comparison to obtain information about methylation and hydroxymethylation modification relative levels. A use of the method is also provided.
摘要翻译： 用于检测核酸中羟基甲基化修饰的方法包括：使核酸糖基化，用MspI消化，将消化的片段两端连接到生物素标记的接头，用NlaIII消化; 使用链霉抗生物素蛋白磁珠捕获消化的片段，以在另一端产生具有生物素标记的接头的片段和另一端的CATG 4-碱基粘性末端，其中这些片段揭示其相邻CCGG位点的修饰信息; 将CATG粘性末端连接到含有MmeI或Ecop15I的识别位点的接头，用相应的限制性内切核酸酶消化以产生可以揭示其相邻CCGG位点的修饰信息的短序列片段; 并进行标签号比较以获得关于甲基化和羟甲基化修饰相关水平的信息。 还提供了该方法的使用。
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