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Comparison of several procedures for plasmid profile determination in Escherichia coli

1995; Elsevier BV; Volume: 22; Issue: 1 Linguagem: Inglês

10.1016/0167-7012(94)00070-n

1872-8359

Anne Bertin,

Bacteriophages and microbial interactions

A comparison has been made between the plasmid content of Escherichia coli containing reference plasmids, as well as of Enterotoxigenic Escherichia coli (ETEC) after several common plasmid extraction procedures and agarose gel electrophoresis. The results were not equivalent for the various extraction procedures. Procedure I (Kado and Liu, 1981) determine both small (here referred to as plasmids migrating before the chromosome) and large (here referred to as plasmids migrating after the chromosome) plasmids of from 1.3 to 144 MDa. However, many chromosomes were present and this could mask plasmids migrating at this level. Additionally some plasmid DNA bands migrating after the chromosome could be weak or fuzzy. Procedures II (Holmes and Quigley, 1981) and III (procedure II with an additional phenol/chloroform extraction stage, Sambrook, 1989) allowed extraction of only part of the plasmids, and often several DNA bands were identified for one reference plasmid giving evidence of the presence of forms other than CCC DNA. The addition of a phenol/chloroform extraction stage to procedure II gave improved determination of small plasmids and permitted determination of some more of the large plasmíds. Procedure IV (Birnboim and Doly, 1979) permitted thin and easily visible DNA bands migrating before or after the chromosome to be obtained, and few chromosomes were present. Some additional DNA bands migrating after the chromosome were also seen or were more visible. As with procedures II and III the largest plasmids Rts1 (120 MDa) and (TP116 143.6 MDa) were not present. When the plasmid sizes of ETEC strains were determined by either procedure I or IV, the values obtained for the large plasmids did not generally differ significantly, except for the higher plasmids of strains B41∗, B117 and 1676, which were up to 12.3% higher with procedure IV. The small plasmids were probably better determined by procedure IV, as shown by the weaker standard errors regularly obtained. The molecular weights of some small plasmids differed notably according to whether they were estimated by either procedure I or IV, suggesting the presence of different plasmids or conformations of the same plasmid. These results demonstrated the importance of considering different possible results when deciding which plasmid extraction procedure is to be used for a specific purpose.