公开(公告)号：US20100291633A1

公开(公告)日：2010-11-18

申请号：US12525905

申请日：2008-02-05

申请人： Thorsten Selmer ,  Olaf Pinkenburg

发明人： Thorsten Selmer ,  Olaf Pinkenburg

IPC分类号： C12P19/34 ,  C12N15/63

CPC分类号： C12N15/66 ,  C12N15/10 ,  C12N15/64

摘要： The present invention refers to methods of (sub)cloning at least one nucleic acid molecule of interest. One embodiment relates to a method of (sub)cloning at least one nucleic acid molecule of interest comprising a) providing at least one (replicable) Entry vector into which the at least one nucleic acid molecule of interest is to be inserted, wherein the at least one Entry vector carries two recognition sites for at least one first type IIS and/or type IIS like restriction endonuclease and wherein said at least one nucleic acid molecule of interest can be excised from the at least one Entry vector at two combinatorial sites with one (same) or more (different) cohesive ends that are formed by the at least one first type IIS or type IIS like restriction endonuclease, and b) providing an Acceptor vector, into which the at least one nucleic acid molecule of interest is transferred from the at least one Entry vector carrying the at least one nucleic acid molecule of interest, wherein said Acceptor vector comprises at least one recognition site for at least one second type IIS restriction endonuclease and/or at least one recognition sites for at least one type IIS like restriction endonuclease, and wherein said Acceptor vector provides two combinatorial sites identical to the two combinatorial sites present in the Entry vector. The inventions also relates respective cloning vector and kits.

摘要翻译： 本发明涉及（亚）克隆至少一种目的核酸分子的方法。 一个实施方案涉及（亚）克隆至少一种感兴趣的核酸分子的方法，包括：a）提供至少一个（可复制）Entry载体，其中所述至少一个目标核酸分子将被插入其中，其中在 至少一个Entry载体携带用于至少一种第一类型IIS和/或类型IIS的限制性内切核酸酶的两个识别位点，并且其中可以在两个组合位点从所述至少一个入口载体中切除所述至少一个目的核酸分子，其中一个 （相同）或更多（不同的）粘性末端，其由至少一种第一类型的IIS或类型的IIS类似的限制性内切核酸酶形成，以及b）提供受体载体，其中所述至少一种目标核酸分子从 所述至少一个Entry载体携带所述至少一个感兴趣的核酸分子，其中所述受体载体包含至少一个第二类型IIS限制性末端的识别位点 核酸酶和/或至少一种类型的IIS类似限制性内切核酸酶的识别位点，并且其中所述受体载体提供与入口载体中存在的两个组合位点相同的两个组合位点。 本发明还涉及各自的克隆载体和试剂盒。