Dhaese P, De Greve H, Decraemer H, Schell J, Van Montagu M
Nucleic Acids Res. 1979 Dec 11;7(7):1837-49. doi: 10.1093/nar/7.7.1837.
A procedure is presented, that has allowed the rapid assignment of transposon Tn1 and Tn7 insertion sites in the large (130 Md) nopaline Ti-plasmid pTiC58, to specific restriction enzyme fragments. Total bacterial DNA is isolated from Agrobacterium tumefaciens strain C58 mutants that carry a transposon in their Ti-plasmid, and digested with an appropriate restriction endonuclease. The fragments are separated on an agarose gel, denatured and transferred to nitrocellulose filters. These are hybridized against purified wild type pTiC58, or against segments of PTiC58, cloned in E. coli using pBR322 as a vector plasmid. DNA sequences homologous to the probe are detected by autoradiography, thus generating a restriction enzyme pattern of the plasmid from a digest of total bacterial DNA. Mutant fragments can be readily identified by their different position compared to a wild type reference. This protocol eliminates the need to separate the large plasmid from chromosomal DNA for every mutant. In principle, it can be applied to the restriction enzyme analysis of insertion or deletion mutants in any plasmid that has no extensive homology with the chromosome.
本文介绍了一种方法，该方法能够将转座子Tn1和Tn7在大的（130 Md）胭脂碱型Ti质粒pTiC58中的插入位点快速定位到特定的限制性内切酶片段上。从在其Ti质粒中携带转座子的根癌土壤杆菌菌株C58突变体中分离出总细菌DNA，并用合适的限制性内切酶进行消化。片段在琼脂糖凝胶上分离，变性后转移到硝酸纤维素滤膜上。将这些滤膜与纯化的野生型pTiC58杂交，或与使用pBR322作为载体质粒在大肠杆菌中克隆的PTiC58片段杂交。通过放射自显影检测与探针同源的DNA序列，从而从总细菌DNA的消化物中生成质粒的限制性内切酶图谱。与野生型对照相比，突变片段可通过其不同位置轻松识别。该方案无需为每个突变体从染色体DNA中分离出大质粒。原则上，它可应用于对任何与染色体无广泛同源性的质粒中的插入或缺失突变体进行限制性内切酶分析。
