Clarke L, Carbon J
Cell. 1976 Sep;9(1):91-9. doi: 10.1016/0092-8674(76)90055-6.
Using the poly(dA-dT) "connector" method (Lobbanand Kaiser, 1973), a population of annealed hybrid circular DNAs was constructed in vitro; each hybrid DNA circle contained one molecule of poly(dT)-tailed Col El-DNA (LRI) annealed to any one of a collection of poly(dA)-tailed linear DNA fragments, produced originally by shearing total E. coli DNA to an average size of 8.5 x 10(6) daltons. This annealed DNA preparation (12 mug) was used to transform an F+ recA E. coli strain (JA200), selecting transformants by their resistance to colicin El. A collection or "bank" pf pver 2000 colicin El-resistant clones was thereby obtained, 70% of which were shown to contain hybrid Col El DNA (E. coli) plasmids. This colony bank is large enough to include hybrid plasmids representative of the entire E. coli genome. Individual plasmids have been readily identified by replica mating the collection onto plates seeded with cultures of various F- auxotrophic recipients, selecting for complementation of the auxotrophic markers by F-mediated transfer of hybrid plasmids to the F- recipients. In this manner, over 80 hybrid Col El-DNA (E. coli), plasmid-bearing clones have been identified in the colony bank, and about 40 known E. coli genes have been tentatively assigned to these various plasmids. The hybrid plasmids are transferred efficiently from F+ donors to appropriate F- recipients. The use of this method to establish similar colony banks in E. coli containing hybrid plasmids representative of various simple eucaryotic genomes is discussed.
利用聚（dA-dT）“连接子”方法（洛班和凯泽，1973年），在体外构建了一批退火杂交环状DNA；每个杂交DNA环包含一个聚（dT）尾的Col E1-DNA（LRI）分子，该分子与一组聚（dA）尾线性DNA片段中的任何一个退火，这些线性DNA片段最初是通过将总大肠杆菌DNA剪切成平均大小为8.5×10⁶道尔顿而产生的。将这种退火的DNA制剂（12微克）用于转化F⁺ recA大肠杆菌菌株（JA200），通过对大肠杆菌素E1的抗性筛选转化体。由此获得了一个超过2000个抗大肠杆菌素E1克隆的集合或“文库”，其中70%被证明含有杂交Col E1 DNA（大肠杆菌）质粒。这个菌落文库足够大，足以包含代表整个大肠杆菌基因组的杂交质粒。通过将该集合复制接种到接种有各种F⁻营养缺陷型受体培养物的平板上，通过F介导的杂交质粒向F⁻受体转移来选择营养缺陷型标记的互补，从而很容易鉴定出单个质粒。通过这种方式，在菌落文库中已经鉴定出80多个携带杂交Col E1-DNA（大肠杆菌）质粒的克隆，并且大约40个已知的大肠杆菌基因已被初步分配到这些不同的质粒上。杂交质粒能有效地从F⁺供体转移到合适的F⁻受体。本文讨论了使用这种方法在大肠杆菌中建立类似的菌落文库，其中包含代表各种简单真核基因组的杂交质粒。
