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MuA-mediated in vitro cloning of circular DNA: transpositional autointegration and the effect of MuB

Author:
Pulkkinen, Elsi, Haapa-Paananen, Saija, Savilahti, Harri
Source:
Molecular genetics and genomics 2016 v.291 no.3 pp. 1181-1191
ISSN:
1617-4615
Subject:
Escherichia coli, bacteriophages, genomics, molecular cloning, plasmids, replication origin, stoichiometry, transposons
Abstract:
Transposons provide useful tools for genetics and genomics studies, as they can be modified easily for a variety of purposes. In this study, a strategy to clone circular DNA was developed on the basis of an efficient Mu in vitro transposition reaction catalyzed by MuA transposase. The transposon used contains a selectable marker as well as an origin of replication, and in vitro integration of the transposon into circular DNA generates a plasmid that can replicate in E. coli. We show that the substrate stoichiometry plays an important role in the profile of intermolecular versus intramolecular transposition reaction products. Increasing the relative amount of target DNA reduced the frequency of intramolecular products that are non-productive with regard to the developed cloning application. Such autointegration was also reduced in the reactions containing phage Mu-encoded MuB, indicating that this protein can be used for cloning in combination with MuA, and it is particularly useful with a limited amount of target DNA. The developed strategy can now be utilized to clone DNA circles regardless of their origin as long as their size is not prohibitive for transformation.
Agid:
5207448