Publication: Cloning of pre-mir-367 coding sequence by homologous recombination and DNA ligation methods
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Date
2026-01
Authors
Wun, Lim Yin
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Abstract
MicroRNAs (miRNAs) are important post-transcriptional regulators involved in
diverse biological processes. Pre-miRNAs serve as essential intermediates in miRNA
biogenesis, and the construction of pre-miRNA expression vectors is critical for functional
characterisation of specific miRNAs such as pre-miR-367. Plasmid construction in our
laboratory has primarily been performed using restriction-ligation cloning, while
homologous recombination has not yet been applied for assembling plasmid containing
microRNA inserts. This study aimed to construct a recombinant pCMV-MiR plasmid
carrying the pre-miR-367 sequence using two cloning strategies: homologous recombination
and restriction–ligation cloning. Cloning outcomes were verified using colony PCR, agarose
gel electrophoresis, and Sanger sequencing. Homologous recombination produced bacterial
colonies, with initial colony PCR indicating potential recombinant clones; however, Sanger
sequencing confirmed the absence of the pre-miR-367 insert in the recovered plasmids. In
contrast, the restriction–ligation approach did not yield transformant colonies under the
conditions tested. Although a sequence-verified recombinant pCMV-MiR–pre-miR-367
construct was not obtained, this study highlights key technical challenges associated with
cloning small and structurally complex pre-miRNA inserts and provides methodological
insights that may support future optimisation of miRNA expression vector construction.