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1. 发明申请

WO2011154147A1 SYSTEM AND METHOD OF MODULAR CLONING 审中-公开
标题翻译：模块化克隆的系统和方法
公开(公告)号：WO2011154147A1
公开(公告)日：2011-12-15
申请号：PCT/EP2011/002843
申请日：2011-06-09
申请人： ICON GENETICS GMBH , WEBER, Ernst , WERNER, Stefan , ENGLER, Carola , GRUETZNER, Ramona , MARILLONNET, Sylvestre
发明人： WEBER, Ernst , WERNER, Stefan , ENGLER, Carola , GRUETZNER, Ramona , MARILLONNET, Sylvestre
IPC分类号： C12N15/66
CPC分类号： C12N15/66 , C12N15/1093
摘要： System for producing a nucleic acid construct of interest, said system comprising: a set of n entry DNAs numbered 1 to n, n being an integer of at least 2, each of said n entry DNAs comprising in this order: (i) a type lIs restriction endonuclease recognition site followed by the cleavage site thereof; (ii) a sequence portion linking the cleavage site of said recognition site of item (i) with the cleavage site of the recognition site of the following item (iii), and (iii) a cleavage site of a further type Ms restriction endonuclease recognition site followed by the recognition site of said cleavage site; the cleavage sites of the type lIs restriction endonuclease recognition sites of item (iii) of entry DNAs 1 to n-1 are complementary to the cleavage sites of the type lIs restriction endonuclease recognition sites of item (i) of entry DNAs 2 to n, respectively; the cleavage site of the type Ms restriction endonuclease recognition site of item (iii) of entry DNA n is complementary to the cleavage site of the type lIs restriction endonuclease recognition site of item (i) of entry DNA 1 for allowing annealing of complementary single-stranded overhangs formed by restriction at recognition site (i) of entry DNA 1 and at recognition site (iii) of entry DNA n; said system further comprising a destination vector comprising in this order: (I) a type Ms restriction endonuclease recognition site followed by the cleavage site thereof; (II) a vector backbone preferably comprising a selectable marker gene, said vector backbone linking the cleavage sites of said recognition sites of items (I) and the following item (III); (III) a further cleavage site of a type Ms restriction endonuclease recognition site followed by the recognition site of said cleavage site, and (IV) optionally, an insert between the recognition sites of item (III) and item (i); said cleavage sites of items (I) and (III) being different and non-complementary, said recognition sites of items (I) and (III) being preferably recognitions sites of the same endonuclease.
摘要翻译：用于产生感兴趣的核酸构建体的系统，所述系统包括：一组n至n的n个入口DNA，n为至少2的整数，所述n个入口DNA中的每一个按以下顺序包括：（i） lIs限制性内切核酸酶识别位点，然后是其切割位点; （ii）将项目（i）的所述识别位点的切割位点与下述（iii）的识别位点的切割位点连接的序列部分，和（iii）另外类型的Ms限制性内切核酸酶识别的切割位点其后是所述切割位点的识别位点; 入口DNA1至n-1项目（ⅲ）的III型限制性内切核酸酶识别位点的切割位点与条目DNA2至n的第（i）项的第II型限制性内切核酸酶识别位点的切割位点互补，分别; 入口DNA n项目（iii）的Ms限制性内切核酸酶识别位点的切割位点与用于允许互补单链DNA退火的入口DNA 1的第（i）项的I型限制性内切核酸酶识别位点的切割位点互补，在入口DNA 1的识别位点（i）和进入DNA n的识别位点（iii）处形成的疏水突出物; 所述系统还包括目的载体，其包含以下顺序：（I）Ms型限制性内切核酸酶识别位点，其后是其切割位点; （II）优选包含选择性标记基因的载体骨架，所述载体骨架连接项目（I）的所述识别位点的切割位点和下列项目（III）; （III）另外的Ms限制性内切核酸酶识别位点的切割位点，随后是所述切割位点的识别位点，和（IV）任选地，（III）和（i）项的识别位点之间的插入片段; 所述项目（I）和（III）的切割位点是不同的和不相互补充的，所述项目（I）和（III）的所述识别位点优选为相同核酸内切酶的识别位点。

2. 发明申请

WO2010040531A3 PROCESS OF CLEAN CLONING 审中-公开
标题翻译：清洁过程
公开(公告)号：WO2010040531A3
公开(公告)日：2010-07-01
申请号：PCT/EP2009007237
申请日：2009-10-08
申请人： ICON GENETICS GMBH , MARILLONNET SYLVESTRE , WERNER STEFAN , ENGLER CAROLA , KANDZIA ROMY , THIEME FRANK , WEBER ERNST
发明人： MARILLONNET SYLVESTRE , WERNER STEFAN , ENGLER CAROLA , KANDZIA ROMY , THIEME FRANK , WEBER ERNST
IPC分类号： C12N15/09 , C12N15/10
CPC分类号： C12N15/64 , C12N15/66
摘要： A process of inserting a nucleic acid sequence of interest into an acceptor nucleic acid, comprising the following steps: amplifying by PCR a DNA comprising in the following order a sequence segment U, a nucleic acid sequence segment of known nucleotide sequence K2 and a nucleic acid sequence segment of known sequence K3 using a forward primer defining a first end of the amplified DNA and a reverse primer defining a second end of the amplified DNA, said reverse primer terminating at its 3'-end in a nucleotide sequence of nucleic acid sequence segment K3; treating the linear double-stranded DNA molecules contained in the PCR product obtained in the previous step with an exonuclease to obtain a single-stranded overhang at the first end of the DNA and a single-stranded overhang comprising nucleic acid segments K2 and K3 at the second end of the DNA; and annealing the product of the previous step to a linearized double-stranded acceptor nucleic acid having at a first end thereof a single-stranded overhang complementary to the single-stranded overhang of the first end of the DNA and at a second end thereof a single- stranded overhang complementary to the single-stranded sequence segment K2 of the second end of the DNA.
摘要翻译：包括以下步骤：通过PCR扩增包含以下顺序的DNA序列的已知核苷酸序列K2的核酸序列片段和核酸的方法已知序列K3的序列片段使用限定扩增DNA第一末端的正向引物和限定扩增DNA第二末端的反向引物，所述反向引物在其核酸序列片段的核苷酸序列的3'末端终止 K3; 用外切核酸酶处理前一步骤中获得的PCR产物中包含的线性双链DNA分子，以在DNA的第一端获得单链突出端，并且在第一端包含核酸片段K2和K3的单链突出端 DNA的第二端; 并将前述步骤的产物退火到线性双链受体核酸，其在第一端具有与DNA的第一末端的单链突出部互补的单链突出端，并且在其第二端具有单链突出端 - 与DNA的第二末端的单链序列片段K2互补的双链突出端。

3. 发明申请

WO2010040531A2 PROCESS OF CLEAN CLONING 审中-公开
标题翻译：清洁克隆过程
公开(公告)号：WO2010040531A2
公开(公告)日：2010-04-15
申请号：PCT/EP2009/007237
申请日：2009-10-08
申请人： ICON GENETICS GMBH , MARILLONNET, Sylvestre , WERNER, Stefan , ENGLER, Carola , KANDZIA, Romy , THIEME, Frank , WEBER, Ernst
发明人： MARILLONNET, Sylvestre , WERNER, Stefan , ENGLER, Carola , KANDZIA, Romy , THIEME, Frank , WEBER, Ernst
IPC分类号： C12N15/09 , C12N15/10
CPC分类号： C12N15/64 , C12N15/66
摘要： A process of inserting a nucleic acid sequence of interest into an acceptor nucleic acid, comprising the following steps: amplifying by PCR a DNA comprising in the following order a sequence segment U, a nucleic acid sequence segment of known nucleotide sequence K2 and a nucleic acid sequence segment of known sequence K3 using a forward primer defining a first end of the amplified DNA and a reverse primer defining a second end of the amplified DNA, said reverse primer terminating at its 3'-end in a nucleotide sequence of nucleic acid sequence segment K3; treating the linear double-stranded DNA molecules contained in the PCR product obtained in the previous step with an exonuclease to obtain a single-stranded overhang at the first end of the DNA and a single-stranded overhang comprising nucleic acid segments K2 and K3 at the second end of the DNA; and annealing the product of the previous step to a linearized double-stranded acceptor nucleic acid having at a first end thereof a single-stranded overhang complementary to the single-stranded overhang of the first end of the DNA and at a second end thereof a single- stranded overhang complementary to the single-stranded sequence segment K2 of the second end of the DNA.
摘要翻译：将感兴趣的核酸序列插入受体核酸中的方法，包括以下步骤：通过PCR扩增包含以下顺序的DNA的序列：序列区段U，核酸序列片段使用限定扩增的DNA的第一末端的正向引物和限定扩增的DNA的第二末端的反向引物的已知核苷酸序列K2和已知序列K3的核酸序列区段，所述反向引物在其3'末端终止于核酸序列片段K3的核苷酸序列; 用外切核酸酶处理在前一步骤中获得的PCR产物中所含的线性双链DNA分子，以在DNA的第一末端获得单链突出端和单链突出端，所述单链突出端包含位于第一端的核酸片段K2和K3 DNA的第二末端; 和将前一步骤的产物退火成线性化双链受体核酸，所述线性化双链受体核酸在其第一末端具有与DNA第一末端的单链突出端互补的单链突出端，并且在其第二末端具有单一突出端 - 与DNA第二末端的单链序列片段K2互补的多余突出端。

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