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1. 发明申请

US20010031483A1 Primer-mediated polynucleotide synthesis and manipulation techniques 审中-公开
标题翻译：引物介导的多核苷酸合成和操作技术
公开(公告)号：US20010031483A1
公开(公告)日：2001-10-18
申请号：US09795965
申请日：2001-02-28
申请人： Stratagene
发明人： Joseph A. Sorge , Kerstien A. Padgett
IPC分类号： C12P001/00 , C07K001/00 , C07K014/00 , C07K017/00 , C12Q001/68
CPC分类号： C12N15/66 , C12N15/10 , C12Q1/686 , C12Q2525/131 , C12Q2525/117
摘要： The invention provides improved techniques for conveniently manipulating polynucleotides of interest without the need to rely upon the presence of naturally occurring restriction sites. Additionally, using the methods and primers of the invention, one may synthesize a polynucleotide of interest in a form which is easily and directionally cloned into a DNA sequence of choice without necessarily introducing extraneous nucleotides in the final polynucleotide product. The methods of the invention employ releasable primers that comprise a recognition site for a releasing enzyme joined to a region for annealing to the polynucleotide template of interest. Polynucleotide sequences of interest are synthesized using one or more synthesis primers, wherein at least one of the primers is a releasable primer. After synthesis, the synthesis product is cleaved by a releasing enzyme. In a preferred embodiment of the invention, inhibitory base analogs are incorporated in the synthesis product to protect against the formation of unwanted internal cleavage products. In another embodiment of the invention, at least one of the releasable primers is bound to an immobilizing solid phase support so as to produce immobilized synthesis products that may be conveniently released by a releasing enzyme. Another aspect of the invention is to provide releasable primers and kits for performing the subject methods. Typically, such kits may comprise a releasing enzyme and one or more reagents for performing a polynucleotide synthesis reaction, preferably a cyclic amplification reaction.
摘要翻译：本发明提供了用于方便地操作感兴趣的多核苷酸的改进的技术，而不需要依赖天然存在的限制性位点的存在。另外，使用本发明的方法和引物，人们可以以容易且定向克隆到选择的DNA序列中的形式合成感兴趣的多核苷酸，而不必在最终多核苷酸产物中引入外来核苷酸。本发明的方法使用可释放的引物，其包含连接到用于退火的感兴趣的多核苷酸模板的区域的释放酶的识别位点。使用一种或多种合成引物合成感兴趣的多核苷酸序列，其中至少一种引物是可释放引物。合成后，合成产物被释放酶切割。在本发明的优选实施方案中，将抑制性碱基类似物掺入合成产物中以防止形成不需要的内部裂解产物。在本发明的另一个实施方案中，至少一个可释放的引物与固定的固相载体结合，以产生可通过释放酶方便地释放的固定的合成产物。本发明的另一方面是提供用于实施本发明方法的可释放的底漆和试剂盒。通常，这样的试剂盒可以包含释放酶和用于进行多核苷酸合成反应的一种或多种试剂，优选循环扩增反应。

2. 发明申请

US20020197642A1 Primer-mediated polynucleotide synthesis and manipulation techniques 审中-公开
标题翻译：引物介导的多核苷酸合成和操作技术
公开(公告)号：US20020197642A1
公开(公告)日：2002-12-26
申请号：US10211341
申请日：2002-08-05
申请人： Stratagene
发明人： Joseph A. Sorge , Kerstien A. Padgett
IPC分类号： C12Q001/68 , C12P019/34
CPC分类号： C12N15/66 , C12N15/10 , C12Q1/686 , C12Q2525/131 , C12Q2525/117
摘要： The invention provides improved techniques for conveniently manipulating polynucleotides of interest without the need to rely upon the presence of naturally occurring restriction sites. Additionally, using the methods and primers of the invention, one may synthesize a polynucleotide of interest in a form which is easily and directionally cloned into a DNA sequence of choice without necessarily introducing extraneous nucleotides in the final polynucleotide product. The methods of the invention employ releasable primers that comprise a recognition site for a releasing enzyme joined to a region for annealing to the polynucleotide template of interest. Polynucleotide sequences of interest are synthesized using one or more synthesis primers, wherein at least one of the primers is a releasable primer. After synthesis, the synthesis product is cleaved by a releasing enzyme. In a preferred embodiment of the invention, inhibitory base analogs are incorporated in the synthesis product to protect against the formation of unwanted internal cleavage products. In another embodiment of the invention, at least one of the releasable primers is bound to an immobilizing solid phase support so as to produce immobilized synthesis products that may be conveniently released by a releasing enzyme. Another aspect of the invention is to provide releasable primers and kits for performing the subject methods. Typically, such kits may comprise a releasing enzyme and one or more reagents for performing a polynucleotide synthesis reaction, preferably a cyclic amplification reaction.
摘要翻译：本发明提供了用于方便地操作感兴趣的多核苷酸的改进的技术，而不需要依赖天然存在的限制性位点的存在。另外，使用本发明的方法和引物，人们可以以容易且定向克隆到选择的DNA序列中的形式合成感兴趣的多核苷酸，而不必在最终多核苷酸产物中引入外来核苷酸。本发明的方法使用可释放的引物，其包含连接到用于退火的感兴趣的多核苷酸模板的区域的释放酶的识别位点。使用一种或多种合成引物合成感兴趣的多核苷酸序列，其中至少一种引物是可释放引物。合成后，合成产物被释放酶切割。在本发明的优选实施方案中，将抑制性碱基类似物掺入合成产物中以防止形成不需要的内部裂解产物。在本发明的另一个实施方案中，至少一个可释放的引物与固定的固相载体结合，以产生可通过释放酶方便地释放的固定的合成产物。本发明的另一方面是提供用于实施本发明方法的可释放的底漆和试剂盒。通常，这样的试剂盒可以包含释放酶和用于进行多核苷酸合成反应的一种或多种试剂，优选循环扩增反应。

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