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    • 3. 发明公开
    • 맞춤형 멀티사이트 조합 어셈블리
    • 定制多地点组合装配
    • KR1020100045494A
    • 2010-05-03
    • KR1020107003986
    • 2008-07-31
    • 비피 코포레이션 노쓰 아메리카 인코포레이티드
    • 탄주퀴우
    • C12N15/10C12P19/34C12Q1/68
    • C12P19/34C12N15/102C12N15/1024C12R1/19
    • The present invention provides a novel method of producing a plurality of modified polynucleotides having different combinations of various mutations at multiple sites by a tailored multi-site combinatorial assembly, comprising adding at least two or at least three primers to a double stranded template polynucleotide in a single reaction mixture, wherein the primers are not overlapping, and wherein each of the primers comprise at least one mutation different from the other primers, wherein at least one primer is a forward primer that can anneal to a minus strand of the template and at least one primer is a reverse primer that can anneal to a plus strand of the template, and subjecting the reaction mixture to a polymerase extension reaction to yield a plurality of extended modified polynucleotides from the at least three primers. The method can be performed without employing a ligation step prior to transforming the extended modified polynucleotides into a cell. The plurality of extended modified polynucleotides can be treated with an enzyme for destroying the template polynucleotide prior to transforming in to the cell.
    • 本发明提供了一种通过定制的多位点组合组合产生多个具有多个位点的不同突变组合的多个修饰的多核苷酸的新方法,包括向双链模板多核苷酸中添加至少两个或至少三个引物 单反应混合物,其中引物不重叠,并且其中每个引物包含与其它引物不同的至少一个突变,其中至少一个引物是可以与模板的负链退火的至少一个引物,并且至少 一个引物是可以与模板的正链退火的反向引物,并且使反应混合物进行聚合酶延伸反应,以从至少三种引物产生多个延伸的修饰的多核苷酸。 在将延伸的修饰的多核苷酸转化成细胞之前,可以不使用连接步骤进行该方法。 多个延伸的修饰的多核苷酸可以在用于在转化到细胞之前破坏模板多核苷酸的酶处理。
    • 4. 发明授权
    • 맞춤형 멀티사이트 조합 어셈블리
    • 定制多地点组合装配
    • KR101685712B1
    • 2016-12-12
    • KR1020107003986
    • 2008-07-31
    • 비피 코포레이션 노쓰 아메리카 인코포레이티드
    • 탄주퀴우
    • C12N15/10C12P19/34C12Q1/68
    • C12P19/34C12N15/102C12N15/1024
    • 본발명은, 단일반응혼합물의이중가닥주형폴리뉴클레오티드에 2종이상또는 3종이상의프라이머를첨가하는단계로서, 상기프라이머는중복되지않고상기프라이머각각은다른프라이머와상이한하나이상의돌연변이를포함하며, 1종이상의프라이머는상기주형의마이너스가닥에어닐링할수 있는정방향프라이머이고, 1종이상의프라이머는상기주형의플러스가닥에어닐링할수 있는역방향프라이머인단계, 및상기반응혼합물에대해폴리머라제연장반응을실시하여, 상기 3종이상의프라이머로부터복수의연장된변형폴리뉴클레오티드를생성하는단계를포함하는, 맞춤형멀티사이트조합어셈블리에의해복수의위치에각종돌연변이의다양한조합을갖는복수의변형폴리뉴클레오티드를제조하기위한신규한방법을제공한다. 이방법은상기연장된변형폴리뉴클레오티드로세포를형질전환시키기전에결찰단계를이용하지않고서수행될수 있다. 세포를형질전환시키기전에주형폴리뉴클레오티드를파괴하기위해상기복수의연장된변형폴리뉴클레오티드를효소로처리할수 있다.
    • 本发明提供了一种通过定制的多位点组合组合产生多个具有多个位点的不同突变组合的多个修饰的多核苷酸的新方法,包括向双链模板多核苷酸中添加至少两个或至少三个引物 单反应混合物,其中引物不重叠,并且其中每个引物包含与其它引物不同的至少一个突变,其中至少一个引物是可以与模板的负链退火的至少一个引物,并且至少 一个引物是可以与模板的正链退火的反向引物,并且使反应混合物进行聚合酶延伸反应,以从至少三种引物产生多个延伸的修饰的多核苷酸。 在将延伸的修饰的多核苷酸转化成细胞之前,可以不使用连接步骤进行该方法。 多个延伸的修饰的多核苷酸可以在用于在转化到细胞之前破坏模板多核苷酸的酶处理。
    • 6. 发明公开
    • HARS36서열을 이용하여 효모 한세눌라 폴리모르파균주에서 라이브러리를 제조하는 방법 및 상기 방법에의하여 제조된 라이브러리
    • 使用HARS36序列和由其构建的图书馆在汉森多聚体中构建图书馆的方法
    • KR1020050029374A
    • 2005-03-28
    • KR1020030065622
    • 2003-09-22
    • 한국생명공학연구원
    • 최의성손정훈김소영배정훈
    • C12N15/81
    • C12N15/1024C12N15/815
    • A method for construction of a library in Hansenula polymorpha using a HARS36 sequence and the library constructed by the same method are provided, thereby constructing a useful gene library having high transformation efficiency and uniformed copy number in Hansenula polymorpha without Escherichia coli. The method for construction of the library in Hansenula polymorpha using the HARS36 sequence comprises the steps of: (1) preparing an insertion gene fragment comprising the HARS36 sequence having the nucleotide sequence of SEQ ID NO:1 of which total or portion sequence is exposed to the 5'- or 3'-terminal of the insertion gene fragment and a target expression gene; (2) a receiving gene fragment comprising a selection marker gene, a promoter for the selection marker gene, and the HARS36 sequence having the nucleotide sequence of SEQ ID NO:1 of which total or portion sequence is exposed to the 5'- or 3'-terminal of the receiving gene fragment; (3) transforming Hansenula polymorpha with both gene fragments of steps (1) and (2) to prepare a transformant; and (4) culturing the transformant.
    • 提供了使用HARS36序列构建汉逊酵母(Hansenula polymorpha)的文库的方法以及通过相同方法构建的文库,由此构建了不含大肠埃希氏菌的多形汉逊酵母中具有高转化效率和均匀拷贝数的有用的基因文库。 使用HARS36序列构建多形汉逊酵母的文库的方法包括以下步骤:(1)制备插入基因片段,其包含具有序列号1的核苷酸序列的HARS36序列,其全部或部分序列暴露于 插入基因片段的5'-或3'端和靶表达基因; (2)包含选择标记基因,选择标记基因的启动子和具有SEQ ID NO:1的核苷酸序列的HARS36序列的接收基因片段,其全部或部分序列暴露于5'-或3 接收基因片段的末端; (3)用步骤(1)和(2)的两个基因片段转化多形汉逊酵母以制备转化体; 和(4)培养转化体。