Universal Restriction Site-Free Cloning Method Using Chimeric Primers
Cloning of PCR products in order to express native proteins, to create chimeric genes, or to study interactions between DNA and proteins requires a method that allows the insertion of the amplified DNA fragment into a vector in a defined position and orientation. Neither the sequence of the DNA fragment nor that of the vector should be changed by the procedure. Routinely used methods such as restriction enzyme and T-A cloning require specific DNA sequences and are, therefore, not always suitable for the above tasks. Although several other methods have been developed to circumvent this problem (e.g., enzymefree cloning,1 heterostagger cloning,2 autosticky PCR cloning,3 and restriction site-free cloning4), their drawbacks may hamper them from being used widely.