发明名称 |
VERFAHREN ZUR SAUBEREN KLONIERUNG |
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;
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; and
transforming the reaction product obtained in the previous step into a host cell. |
申请公布号 |
AT512219(T) |
申请公布日期 |
2011.06.15 |
申请号 |
AT20080017649T |
申请日期 |
2008.10.08 |
申请人 |
ICON GENETICS GMBH |
发明人 |
MARILLONNET, SYLVESTRE;ENGLER, CAROLA;KANDZIA, ROMY;THIEME, FRANK;WEBER, ERNST |
分类号 |
C12N15/09;C12N15/10 |
主分类号 |
C12N15/09 |
代理机构 |
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代理人 |
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主权项 |
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地址 |
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