Universal restriction site-free cloning method using chimeric primers

A universal restriction site-free cloning method has been developed to precisely insert a DNA fragment into a vector at any desired location without altering any nucleotide(s) in either the, DNA fragment or the vector The technique employs two pairs of chimeric primers, each containing a ribonucleotide. One pair of primers is used to amplify a target DNA fragment and another is used to prepare a linear vector The ribonucleotide is used as a specific site for cleavage promoted by rare-earth metal ions such as La3+ or Lu3+. Therefore, blunt-ended PCR products can be converted into a dsDNA with single-stranded 3' overhangs for efficient ligation. The primers are designed so that both the target DNA fragment and vector PCR products create defined 3' overhangs to permit the formation of a seamless plasmid during the subsequent ligation. This method has been used successfully to clone the E. coli gene coding for peptidyl-tRNA hydrolase.