aerospace and mechanical engineering pdf

TA cloning has been used in direct cloning of PCR products in molecular biology laboratories for over
twenty years [1]. This cloning method does not require restriction enzyme digestion of target DNA fragments and usually allows blue-white screening of recombinant plasmids. Addition of an extra adenosine residue by the terminal transferase activity of thermo-stable DNA polymerases (e. g., Taq
DNA polymerase) is the basis of TA cloning method [2, 3, 4]. Although TA cloning still remains popular for its performance in efficiency, convenience and cost saving, it is not suitable for construction of expression vector due to randomness of direction of inserted DNA fragments.

In this report, we devised a novel cloning method that facilitates directional cloning of PCR products directly. The key of the method is the design of a cloning vector with a deoxythymidine
overhang and a deoxycytidine overhang at two 3′ ends respectively. According to previous observations [3, 5], 3′-terminal deoxyguanosine overhang of PCR products occurs only when the
corresponding primer has a 5′-terminal deoxycytidine residue if the PCR reaction is catalyzed by Taq
DNA polymerase. Therefore, to enable the ligation of PCR products to such a linearized vector, one
PCR primer is designed to possess a 5′-terminal deoxycytidine residue and the other to possess a
5′-terminal non-deoxycytidine residue.

Theoretically, PCR product with a deoxyadenosine overhang at one 3′ end and a deoxyguanosine overhang at the other 3′ end will be present in PCR products generated with such primer pair and Taq DNA polymerase. Obviously, only PCR product of this kind can be ligated to a linear vector with a 3′-T overhang and a 3′-C overhang in the mode of TA cloning at one end and GC cloning at the other, generating recombinant plasmids with unidirectional inserts (Fig. 1).