Abstract
Molecular cloning methods based on primer and overlap-extension PCR are widely used due to their simplicity, reliability, low cost and high efficiency. In this article, an efficient mega primer-mediated (MP) cloning strategy for chimaeragenesis and long DNA fragment insertion is presented. MP cloning is a seamless, restriction/ligation-independent method that requires only three steps: (i) the first PCR for mega primer generation; (ii) the second PCR for exponential amplification mediated by the mega primers and (iii) DpnI digestion and transformation. Most importantly, for chimaeragenesis, genes can be assembled and constructed into the plasmid vector in a single PCR step. By employing this strategy, we successfully inserted four DNA fragments (approximately 500 bp each) into the same vector simultaneously. In conclusion, the strategy proved to be a simple and efficient tool for seamless cloning.
Highlights
Seamless cloning and gene fusion are efficient tools for protein engineering, protein functional studies, protein production, promoter and exon studies and genome manipulation [1]
Inspired by the cloning methods mentioned above, we present an efficient mega primer-mediated (MP) cloning strategy named as MP cloning, which is amenable to both chimaera construction and long DNA fragment insertion
These methods are greatly useful for chimaeragenesis construction; they still have their limitations in barricading HTP-cloning applications
Summary
Seamless cloning and gene fusion are efficient tools for protein engineering, protein functional studies, protein production, promoter and exon studies and genome manipulation [1]. Exponential Megapriming PCR (EMP) cloning [5] and Inverse Fusion PCR cloning (IFPC) [6] obviously improve the size of inserts (up to 2.5 kb) by the introduction of an assistant primer to ensure exponential amplification Both methods require phosphorylation and ligation of the final products from the second round of PCR to ensure efficiency, which adds to labour and cost. Another modified version, Circular Polymerase Extension cloning (CPEC) [7], uses two pairs of primers to amplify target DNA and linearize recipient vectors in two separate PCRs. In the second PCR, the insert (single or hybridized) and vector extends using each other as a template until they extend into a circular plasmid with two nicks. Compared with RF cloning and other derivatives, MP cloning shows a much higher efficiency for longer fragment insertion and fragment assembly
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