Chimeric plasmids

FIGURE 13.2 Foreign DNA sequences can be inserted into plasmid vectors by opening the circnlar plasmid with a restriction endonnclease. The ends of the linearized plasmid DNA are then joined with the ends of a foreign sequence, reclosing the circle to create a chimeric plasmid. 

Seal gaps in chimeric plasmid widi DNA ligase... 

Cohen, S. N., Chang, A. C. Y, Boyer, H. W., and Helling, R. B., 1973. Construction of biologically functional bacterial plasmids in vitro. Proceedings of the National Academy of Sciences U.S.A. 70 3240 — 3244. The classic paper on the construction of chimeric plasmids. 

Hybrid (or chimeric) plasmid. A plasmid that contains DNA from two different organisms. 

Whiteway, M. S. and A, Ahmed, "Recombinational Instability of Chimeric Plasmid in Saccharomyces cerevisiae, " Molecular Cellular Biology (January 1984) 195 - 198. 

The plasmid and foreign DNA, both cleaved by the same restriction enzyme, are mixed together and treated with DNA ligase. Some of the interactions produce chimeric plasmids, containing the foreign DNA integrated into the plasmid DNA. 

Clones that contain the chimeric plasmid are selected by growing the cells in media containing the antibiotics to which the plasmid has genes for resistance. 

Cells containing an appropriate chimeric plasmid are cultured (see Figure 3-30). 

Select cells that contain chimeric plasmids... 

Simonsen, C. and A. Levinson, Analysis of processing and polyadenylation signals of the hepatitis B Virus surface antigen gene by using simian vims 40-Hepatitis B Vims chimeric plasmids. Mol. Cell Biol., 1983, 3, 2250-2258. 

A plasmid, which is a circular DNA molecule separate from the chromosomal DNA, is obtained from bacterial cells such as Escherischia coli and treated with a restriction enzyme to snip the DNA at a specific site (Figure 26.15). The human DNA sequence that codes for the synthesis of insulin is then inserted into the plasmid to give a recombinant DNA molecule. The new DNA is the result of the recombination of DNA from the plasmid plus the sequence that codes for human insulin. The new plasmid is termed a chimeric plasmid because it contains DNA from two sources, bacterial and human. The plasmid is taken up by growing bacterial cells through a process called transformation. The chimeric plasma serves as a cloning vector because it serves as a vehicle to carry the recombinant DNA into E. coli. Transcription and translation of the insulin DNA then occur to produce human insulin. When the cells divide, the plasmids are divided between the daughter cells and they continue to produce clones. Insulin produced by recombinant DNA technology is commercially sold as Humulin. 

Fig.5. In vitro generation of a chimeric plasmid containing foreign DNA. The required sticky ends are created by cleaving both the plasmid and the foreign DNA with the same restriction endonuclease (in this example, EcoRI).

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