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2. 发明公开

EP2951316A2 PRIMER TECHNOLOGY 审中-公开
标题翻译： PRIMERTECHNOLOGIE
公开(公告)号：EP2951316A2
公开(公告)日：2015-12-09
申请号：EP14703318.7
申请日：2014-02-03
申请人： Selvi, Ozan , Orcan, Serkan
发明人： SELVI, Ozan , ORCAN, Serkan , TOKSÖZ, Sila
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6853 , C12N15/1048 , C12P19/34 , C12Q1/6806 , C12Q1/6844 , C12Q2521/319 , C12Q2525/143 , C12Q2525/173 , C12Q2525/191
摘要： The present invention provides a method of nucleic acid manipulation comprising: (i) hybridizing a double stranded primer to the 3' end of a single stranded nucleic acid template, wherein said primer comprises a double stranded region and a single stranded region, wherein the single stranded region is a 3' overhang region and wherein the 3' overhang region enables the double stranded primer to target and hybridize to the 3' end of the nucleic acid template, wherein said single stranded 3' overhang region comprises a degenerate sequence or a sequence comprising universal bases, and wherein the double stranded primer is made up of two separate strands; (ii) at least one round of polymerization using a polypeptide with 5' to 3' DNA polymerization activity, wherein at least the first round of polymerization comprises using a polypeptide with 5' to 3' DNA polymerization activity to carry out a primer extension reaction to synthesise nucleotides in a template dependent manner from the 3' end of the single stranded region of said hybridized double stranded primer; wherein the hybridizing step (i) and at least the first primer extension reaction from the 3' end of the single stranded region of said hybridized double stranded primer in step (ii) takes place without the formation of a phosphodiester bond between the 3' end of the nucleic acid template and the double stranded primer of step (i). Products, kits and compositions suitable for use in such methods are also provided.
摘要翻译：本发明提供了一种核酸操作方法，包括：（i）将双链引物与单链核酸模板的3'末端杂交，其中所述引物包含双链区域和单链区域，其中单链引物双链区是3'突出区，其中3'突出区使得双链引物靶向并与核酸模板的3'末端杂交，其中所述单链3'突出区包含简并序列或序列包括通用碱基，并且其中双链引物由两条单独的链组成; （ii）使用具有5'至3'DNA聚合活性的多肽的至少一轮聚合，其中至少第一轮聚合包括使用具有5'至3'DNA聚合活性的多肽以进行引物延伸反应以模板依赖的方式从所述杂交双链引物的单链区域的3'末端合成核苷酸; 其中所述杂交步骤（i）至少在步骤（ii）中所述杂交双链引物的单链区域的3'末端的第一引物延伸反应进行而不在3'末端之间形成磷酸二酯键的核酸模板和步骤（i）的双链引物。还提供适用于这些方法的产品，试剂盒和组合物。

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