The first recombinant DNA plasmid that combined two different biological activities from different sources and was readily transferred into bacteria was reported in 1973 by the research groups of Stanley Cohen and Herbert Boyer. In this work, the investigators began with the large, naturally occurring circular plasmid R6-5 (∼98,500 bp), which included genes conferring resistance to multiple antibiotics, including tetracycline and kanamycin. Prior to their study, R6-5 had been sheared into fragments, the fragments used to transform E. coli, and tetracycline-resistant colonies selected. A smaller circular plasmid had arisen from that work, pSC101 (∼9,000 bp), which replicated normally and conferred only the tetracycline resistance. The researchers assumed that the smaller plasmid represented a piece of the larger one that had ligated into a smaller circle, which included a replication origin and the tetracycline-resistance gene. A third plasmid had also been generated when plasmid R6-5 was cleaved with EcoRI, the fragments used to transform E. coli, and cells selected for growth on kanamycin. This third plasmid was called pSC102 (∼27,000 bp), and it did not confer resistance to tetracycline.

The ultimate objective was to combine pSC101 with fragments from R6-5 or pSC102 to generate a new plasmid that conferred a demonstrable new biological activity. The investigators first cleaved all three plasmids with EcoRI. The cleavage products were subjected to electrophoresis on an agarose gel, and the DNA fragments were visualized by staining with ethidium bromide (a fluorescent molecule that binds to DNA by intercalating between adjacent base pairs). This generated the pattern in Figure 1. Electrophoresis proceeded left to right, as shown in the figure, so the bands decrease in size from left to right (lane a is pSC102; lane b, R6-5; lane c, pSC101).

The plasmids pSC101 and pSC102 were cleaved with EcoRI, and the fragments from both plasmids were mixed together and treated with DNA ligase. The ligated mixture was then used to transform E. coli, and the cells were grown on plates containing both kanamycin and tetracycline. Colonies appeared, and all of them contained a new plasmid, named pSC105 (∼16,000 bp), that conferred resistance to kanamycin and tetracycline. Next, the plasmids pSC101, pSC102, and pSC105 were cleaved with EcoRI and subjected to gel electrophoresis. The results are shown in Figure 2. Electrophoresis proceeded, and fragment sizes decrease, from left to right (lane a is pSC105; lane b, pSC101 + pSC102; lane c, pSC102; lane d, pSC101).