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

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... l ions such as La 3 + or Lu 3 + . 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.