CLONING VECTORS THAT YIELD HIGH-LEVELS OF SINGLE-STRANDED-DNA FOR RAPID DNA SEQUENCING
CLONING VECTORS THAT YIELD HIGH-LEVELS OF SINGLE-STRANDED-DNA FOR RAPID DNA SEQUENCING
复制标题
DOI：
10.1016/0378-1119(84)90139-2
复制
发表时间：
1984-01-01
期刊：
影响因子：
3.5
通讯作者：
BERMAN, ML
中科院分区：
文献类型：
--
作者：
ZAGURSKY, RJ;BERMAN, ML
将M13噬菌体的起始区和基因间区克隆到pBR 322的PvuII（pZ 145）和AhaIII（pZ 150，pZ 152）位点，构建嵌合质粒载体。在不存在M13噬菌体的情况下，这些质粒像任何其他ColE 1衍生的质粒一样复制，并赋予氨苄青霉素和四环素抗性（青霉素，泰特）。在用M13噬菌体感染后，质粒上存在的病毒起点允许噬菌体指导的质粒复制，并导致M13样颗粒中的单链（ss）质粒DNA的高产率。该ssDNA仅代表质粒链中的一条，可用作通过桑格等人描述的双脱氧测序法进行快速DNA序列测定的底物。由于这些质粒含有完整的pBR 322，基因间区可转移到大多数pBR 322衍生物上以产生ss质粒DNA，而不影响受体质粒用于进一步研究。还构建了pZ 145的缺失衍生物，质粒pZ 146，其不表现出对M13辅助细胞生长的干扰，尽管该质粒被掺入噬菌体颗粒中。该结果证实了基因间区由2个结构域组成的理论：一个结构域是参与噬菌体形态发生的区段，另一个结构域是干扰M13复制的功能起源区。[使用大肠杆菌]。
Chimeric plasmid vectors with the origin and intergenic region from M13 phage cloned into the PvuII (pZ145) and AhaIII (pZ150, pZ152) sites of pBR322 were constructed. In the absence of M13 phage, these plasmids replicate like any other ColE1-derived plasmid and confer ampicilln and tetracycline resistance (Amp, Tet). Upon infection with M13 phage, the viral origin present on the plasmids permits phage-directed plasmid replication and results in high yields of single-stranded (ss) plasmid DNA in M13-like particles. This ssDNA, which represents only 1 of the plasmid strands, is useful as a substrate for rapid DNA sequence determination by the dideoxy sequencing method described by Sanger et al. Since these plasmids contain an intact pBR322, the intergenic region can be transferred onto most pBR322 derivatives to yield ss plasmid DNA without affecting the recipient plasmid for further studies. A deletion derivative of pZ145, plasmid pZ146, that does not exhibit interference with the growth of the M13 helper, although this plasmid is encapsidated into phage particles, was also constructed. This result confirms the theory that the intergenic region consists of 2 domains: one domain being a segment involved in phage morphogenesis and the other being a region of functional origin which interferes with M13 replication. [Escherichia coli was used.].