Directional Restriction Site-Free Insertion of PCR Products into Vectors

The polymerase chain reaction (PCR) technique has proved to be a powerful tool for rapid amplification of DNA fragments of interest during cloning. Insertion of PCR products into suitable vectors in order to construct plasmids for protein expression, or to create chimeric genes or to study interactions between proteins and DNA requires a method that allows the precise insertion of a DNA fragment at a defined position in the vector without altering the surrounding DNA sequence. The commonly used cloning methods, such as blunt-end insertion (1 ,2 ), introduction of restriction sites on both ends of the PCR products (3 ,4 ), T vectors (5 ) and ligation-independent cloning (6 ,7 ) do not usually meet the needs because of the limited cloning sites available on the vector and/or the necessity of the nucleotide change. Hence, the cloning process may become very complicated to insert a PCR product into a defined location on the vector without the change of the nucleotides.