Directional cloning of an oligonucleotide fragment into a single restriction site

Oligonucleotide fragments can be directionally subcloned into vectors at a single restriction site. By using T4 DNA polymerase exonuclease activity to treat vector DNA, single-stranded ends can be generated. The oligonucleotide sequences are designed to have sequence complementary to these single-stranded ends. Through the homologous annealing of oligonucleotides to the treated vector ends, the successfully subcloned molecules forms a circular recombinant DNA that is ready for transformation. There is no sequence restriction at the ends of the DNA fragment. All restriction site ends are accessible to this method. This approach for oligonucleotide fragment insertion and together with our previously described general method of exonuclease induced DNA subcloning provide convenient methods for the construction of recombinant DNA.