MCT cloning: a seamless cloning strategy for inserting DNA fragments

Abstract

This study presents a seamless, efficient and restriction/ligation-independent cloning strategy for inserting large fragments: MCT cloning (based on the generation of Manmade Cohesive Termini). This method requires only four steps: (i) the first parallel PCR for the amplification of complete and truncated insert fragments; (ii) the second parallel PCR for the exponential amplification of linear plasmids with the same sequences and different break sites mediated by the first products and the corresponding reverse primers; (iii) the formation of manmade cohesive termini after DpnI digestion, mixing, denaturation and annealing; and (iv) transformation. By employing this strategy, large fragments (1–4 kbp) can be easily inserted into the pGADT7 vector (∼ 8 kbp).