公开(公告)号：US20040253729A1

公开(公告)日：2004-12-16

申请号：US10811062

申请日：2004-03-25

申请人： Stratagene and Children's Medical Center Corporation

发明人： John C. Bauer ,  Dowain A. Wright ,  Jeffrey Carl Braman ,  Raif S. Geha

IPC分类号： C12Q001/68 ,  C12P019/34 ,  C12N015/87 ,  C12N015/74

CPC分类号： C12N15/102 ,  C12Q1/6806 ,  C12Q1/6827 ,  C12Q1/6853 ,  C12Q2600/156 ,  Y10S435/81 ,  C12Q2525/307 ,  C12Q2521/319 ,  C12Q2525/185 ,  C12Q2521/331 ,  C12Q2521/101 ,  C12Q2531/125

摘要： The invention provides improved methods of introducing site-directed mutations into circular DNA molecules of interest by means of mutagenic primer pairs. The mutagenic primer pairs are also selected so as to be either completely complementary or partially complementary to each other, wherein the mutation site (or sites) is located within the region of complementarity. A mutagenic primer pair is annealed to opposite strands of a circular DNA molecule containing the DNA sequence to be mutagenized. After annealing, first and second mutagenized DNA strands, each incorporating a member of the mutagenic oligonucleotide primer pair is synthesized by a linear cyclic amplification reaction. After the linear cyclic amplification mediated synthesis step is completed, the reaction mixture is treated with a selection enzyme that digests the parental template strands. After the digesting step, a double-stranded circular DNA intermediate is formed. The double-stranded circular DNA intermediates is transformed in suitable competent host cells and closed circular double-stranded DNA corresponding to the parental template molecules, but containing the desired mutation or mutations of interest, may be conveniently recovered from the transformed cells. The invention also provide kits for site-directed mutagenesis in accordance with methods of the present invention.

公开(公告)号：US20030064516A1

公开(公告)日：2003-04-03

申请号：US10229390

申请日：2002-08-26

申请人： Stratagene and Children's Medical Center Corporation

发明人： John C. Bauer ,  Dowain A. Wright ,  Jeffrey Carl Braman ,  Raif S. Geha

IPC分类号： C12P019/34 ,  C12N015/85

CPC分类号： C12N15/102 ,  C12Q1/6806 ,  C12Q1/6827 ,  C12Q1/6853 ,  C12Q2600/156 ,  Y10S435/81 ,  C12Q2525/307 ,  C12Q2521/319 ,  C12Q2525/185 ,  C12Q2521/331 ,  C12Q2521/101 ,  C12Q2531/125

摘要： The invention provides improved methods of introducing site-directed mutations into circular DNA molecules of interest by means of mutagenic primer pairs. The mutagenic primer pairs are also selected so as to be either completely complementary or partially complementary to each other, wherein the mutation site (or sites) is located within the region of complementarity. A mutagenic primer pair is annealed to opposite strands of a circular DNA molecule containing the DNA sequence to be mutagenized. After annealing, first, and second mutagenized DNA strands, each incorporating a member of the mutagenic oligonucleotide primer pair is synthesized by a linear cyclic amplification reaction. After the linear cyclic amplification mediated synthesis step is completed, the reaction mixture is treated with a selection enzyme that digests the parental template strands. After the digesting step, a double-stranded circular DNA intermediate is formed. The double-stranded circular DNA intermediates is transformed in suitable competent host cells and closed circular double-stranded DNA corresponding to the parental template molecules, but containing the desired mutation or mutations of interest, may be conveniently recovered from the transformed cells. The invention also provide kits for site-directed mutagenesis in accordance with methods of the present invention.

摘要翻译： 本发明提供了通过诱变引物对将位点定向突变引入感兴趣的环状DNA分子的改进方法。 诱变引物对也被选择为彼此完全互补或部分互补，其中突变位点（或位点）位于互补区域内。 将诱变引物对退火至包含待诱变的DNA序列的环状DNA分子的相反链。 退火后，通过线性循环扩增反应合成每个引入诱变寡核苷酸引物对的成员的第一和第二诱变DNA链。 在线性循环扩增介导的合成步骤完成后，用消化亲本模板链的选择酶处理反应混合物。 消化步骤后，形成双链环状DNA中间体。 将双链环状DNA中间体转化到合适的感受态宿主细胞中，并且可以从转化的细胞中方便地回收对应于亲本模板分子但含有所需的突变或目标突变的闭合环状双链DNA。 本发明还提供了根据本发明方法进行定点诱变的试剂盒。