Directional cloning of PCR products using exonuclease III

Directional cloning of PCR products is usually carried out through different cohesive ends created by digestion with restriction enzymes. Although this method is generally straightforward, it has disadvantages: if present, internal sites will be cleaved by the restriction enzymes; to ensure efficient cleavage, several extra nucleotides have to be added to the 5' end of primers in addition to those providing the restriction site; several sites (Not I, Xba I, Xho I) have been reported recalcitrant to digestion; (iv) occasionally, restriction enzymes buffers are not compatible and digestions have to be carried out sequentially. 5' Cohesive termini can be created by controlled digestion with T4 DNA polymerase. One method is limited to the combination of palindromes consisting of ATs, or GCs, exclusively, and a second method requires a relatively long 5' add-on sequence. The present method is based on exposing different 5' cohesive ends simultaneously by controlled Exo III digestion.