Direct ligation of PCR products for cloning and sequencing

We are studying the expression of cytokines and T cell receptor variable region beta chain (V beta ) genes, using polymerase chain reaction (PCR) in arthritic joints of mice. To clone and sequence the PCR products rapidly we have developed a protocol which requires no manipulation such as gel purification, polishing the ends with T4 DNA polymerase and phosphorylation with T4 Polynucleotide kinase or use of special vectors. We have found that if the final extension step is omitted from the amplification protocol, the resultant DNA fragment can be ligated directly to M13mp18 vector digested with a restriction enzyme with generates blunt ends. We reasoned that since the PCR amplified material is basically a blunt end, dephosphorylated DNA molecule it can be ligated to a vector cut with either EcoRV or SmaI. The situation in this case would simply be reverse of that normal blunt end cloning where the vector is dephosphorylated while the termini of the target molecule carry the phosphate.