Simple Cloning and DNA Assembly in Escherichia coli by Prolonged Overlap Extension PCR

We developed a simple method (Simple Cloning) for subcloning one, two, or three DNA fragments into any location of a targeted vector without the need for restriction enzyme, ligase, exonuclease, or recombinase. This cloning technology can be applied to a few common Escherichia coli hosts (e.g., BL21(DE3), DH5α, JM109, TOP10). The protocol includes three steps: (a) linear DNA fragments (i.e., the insert DNA and the vector backbone) with two overlap ends were generated by regular high-fidelity PCR, (b) the DNA multimers were generated based on these equimolar DNA templates by using prolonged overlap extension PCR (POE-PCR) without primers added, and (c) the POE-PCR product was transformed to E . coli strains directly. Because positive colony efficiencies are very high, it is not necessary to identify desired clones by using colony PCR. Simple Cloning provides a new cloning and DNA assembly method with great simplicity and flexibility.