THE PUC PLASMIDS, AN M13MP7-DERIVED SYSTEM FOR INSERTION MUTAGENESIS AND SEQUENCING WITH SYNTHETIC UNIVERSAL PRIMERS
THE PUC PLASMIDS, AN M13MP7-DERIVED SYSTEM FOR INSERTION MUTAGENESIS AND SEQUENCING WITH SYNTHETIC UNIVERSAL PRIMERS
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DOI：
10.1016/0378-1119(82)90015-4
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发表时间：
1982-01-01
期刊：
影响因子：
3.5
通讯作者：
MESSING, J
中科院分区：
文献类型：
--
作者：
VIEIRA, J;MESSING, J
构建了一系列含有M13mp7多克隆位点（MCS7）的质粒载体。在这些载体之一中，卡那霉素抗性标记被插入到对称MCS7的中心以产生限制性位点移动元件（RSM）。可以用识别RSM侧翼序列位点的任何限制性酶将抗药性标记物从该载体中切割出来，以产生具有各种粘性或平端的RSM。这些片段可用于具有相容限制性位点的任何靶分子的插入诱变。插入突变体通过其对卡那霉素的抗性来选择。当用Pst I去除耐药标记物时，可以产生小的框内插入。通过改变MCS7的对称结构，形成了两个具有单限制性位点的新MCS。得到的质粒pUC 8和pUC 9允许分别克隆相对于lac启动子具有两个方向的双重消化的限制性片段。这些质粒中克隆的任何DNA的末端序列可使用通用M13引物表征。
A series of plasmid vectors containing the multiple cloning site (MCS7) of M13mp7 was constructed. In one of these vectors a kanamycin resistance marker was inserted into the center of the symmetrical MCS7 to yield a restriction-site-mobilizing element (RSM). The drug resistance marker can be cleaved out of this vector with any of the restriction enzymes that recognize a site of the flanking sequences of the RSM to generate an RSM with various sticky or blunt ends. These fragments can be used for insertion mutagenesis of any target molecule with compatible restriction sites. Insertion mutants are selected by their resistance to kanamycin. When the drug-resistance marker is removed with PstI, a small in-frame insertion can be generated. Two new MCS having single restriction sites were formed by altering the symmetrical structure of MCS7. The resulting plasmids pUC8 and pUC9 allow one to clone doubly digested restriction fragments separately with both orientations in respect to the lac promoter. The terminal sequences of any DNA cloned in these plasmids can be characterized using the universal M13 primers.