Open AccessIn vitro amplification of whole large plasmids via transposon-mediated oriC insertion
Author(s) -
Seia Nara,
Masayuki Su’etsugu
Publication year - 2021
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/btn-2021-0068
Subject(s) - plasmid , transposable element , biology , genetics , origin of replication , dna , rolling circle replication , dna replication , microbiology and biotechnology , circular bacterial chromosome , gene , genome
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.