Protocols for cloning and analysis of blunt-ended PCR-generated DNA fragments.

Methods are presented for the improved yield and analysis of blunt-ended cloning of PCR-generated DNA fragments.(1) We show that Pfu DNA polymerase polishing of Taq DNA polymerase-generated fragments increases the yield and efficiency of cloning.Using a triple primer set consisting of two outside, asymmetrically distanced primers and one fragment-specific primer, one can determine both the presence and orientation of cloned inserts.Application of these methods allows one to generate and clone a fragment in 1 day and analyze putative clones the next, thereby saving a substantial amount of both time and effort. VECTOR PREPARATIONSeveral vectors and derivatives have been created for PCR cloning.These include the standard pBluescript-type multiple cloning sites (e.g., pBluescrip-tlI, pBC, pCR-ScriptAmp, and pCR-ScriptCam) (Stratagene, La Jolla CA) and the abbreviated multiple cloning sites as contained in the pCR-Script Direct plasmids (Stratagene) (2-4~ (see Fig. 1).The abbreviated multiple cloning sites allow the end user to incorporate common restriction enzyme sites into their PCR primer sets without the problem of having the same target sequence occurring in the plasmid vector.It is recommended that the chloramphenicol derivatives be used when subcloning DNA fragments generated from ampicillin-resistance encoding plasmids.This ensures that recombinant colonies after Escherichia coli transformation are not the result of parental plasmid transformation. Bidirectional Cloning VectorsBlunt-ended cloning procedures capture blunted PCR fragments for bidirec-FIGURE 1 Vectors for bidirectional and directional cloning of PCR fragments.Several vectors have been developed for directional and bidirectional cloning.These include derivatives that encode either chloramphenicol or ampicillin resistance with modified multiple cloning sites optimized for specific cloning operations (e.g., general subcloning or protein expression).