In vitro amplification of whole large plasmids via transposon-mediated oriC insertion
Seia Nara1, Masayuki Su'etsugu1
1Department of Life Science, College of Science, Rikkyo University, 3-34-1 Nishi-Ikebukuro, Toshima-ku, Tokyo, 1718501, Japan.
Abstract:
DNA amplification is a fundamental technique in molecular biology. The replication cycle reaction is a new method for amplification of large circular DNA having oriC sequences, which is a replication initiation site of the Escherichia coli chromosome. We here developed a replication cycle reaction-based method useful for amplification of various circular DNAs lacking oriC, even in the absence of any sequence information, via transposon-mediated oriC insertion to the circular DNA template. A 15-kb non-oriC plasmid was amplified from a very small amount of starting DNA (50 fg, 1 fM). The method was also applicable to GC-rich plasmid (69%) or large F-plasmid (230 kb). This method thus provides a powerful tool to amplify various environmental circular DNAs.
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