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    PROCESS OF CLEAN CLONING
    4.
    发明申请
    PROCESS OF CLEAN CLONING 审中-公开
    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

    Applicant: ICON GENETICS GMBH ,  MARILLONNET SYLVESTRE ,  WERNER STEFAN ,  ENGLER CAROLA ,  KANDZIA ROMY ,  THIEME FRANK ,  WEBER ERNST

    Inventor： MARILLONNET SYLVESTRE ,  WERNER STEFAN ,  ENGLER CAROLA ,  KANDZIA ROMY ,  THIEME FRANK ,  WEBER ERNST

    IPC: C12N15/09 ,  C12N15/10

    CPC classification number: C12N15/64 ,  C12N15/66

    Abstract: 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.

    Abstract translation: 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。

    PROCESS OF CLEAN CLONING
    5.
    发明申请
    PROCESS OF CLEAN CLONING 审中-公开
    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

    Applicant: ICON GENETICS GMBH ,  MARILLONNET, Sylvestre ,  WERNER, Stefan ,  ENGLER, Carola ,  KANDZIA, Romy ,  THIEME, Frank ,  WEBER, Ernst

    Inventor： MARILLONNET, Sylvestre ,  WERNER, Stefan ,  ENGLER, Carola ,  KANDZIA, Romy ,  THIEME, Frank ,  WEBER, Ernst

    IPC: C12N15/09 ,  C12N15/10

    CPC classification number: C12N15/64 ,  C12N15/66

    Abstract: 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.

    Abstract translation: 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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