(620cc) Improved Forms of Omega-PCR for DNA Plasmid Construction and Manipulation

Senger, R. S., Virginia Tech

The construction of DNA plasmids for metabolic engineering applications has long been a bottleneck in the discipline.  It is either time-consuming and/or unsuccessful through traditional approaches in molecular biology or it can be cost prohibitive to use commercial cloning kits or custom DNA synthesis.  This is particularly true as one tries to incorporate several genes, regulatory parts, and/or recombination sequences into a single DNA plasmid.  In response to this molecular cloning bottleneck, useful solutions have often resulted in commercial ventures.  Here, we describe the original development of omega-PCR (Ω-PCR) and our contributions of single-stranded and double-stranded versions of this technique (ds-Ω-PCR and ss-Ω-PCR).  We also show how these techniques can be used for low cost, single tube, restriction enzyme-free cloning.  The method is highly reliable and can be completed in less than 48 hours, even for the construction of DNA plasmids consisting of multiple parts.  Here, we demonstrate the technique by inserting, removing, and editing multiple fluorescent reporter genes in a common plasmid backbone.