ABC cloning: An efficient, simple, and rapid restriction/ligase-free method.

DNA cloning remains the primary step before the further investigation of gene function. Restriction enzyme-based cloning methods are still widely used and numerous restriction-free cloning techniques are available as alternatives. Here we describe a PCR-based cloning method named ABC cloning. This method uses PCR to combine three overlapping DNA fragments into a recombinant vector that can be immediately transformed into competent cells. This technique uses only a thermostable DNA polymerase and is more rapid and efficient than previously described methods.