In-frame cloning of large synthetic genes using moderate-size oligonucleotides.

A method is described that facilitates the cloning of large synthetic genes in plasmid vectors such as pUC and pGEM. This protocol uses unpurified synthetic oligonucleotides of moderate length (ca. 50-90 bp) to construct larger DNA molecules by a reiterative, directional cloning procedure. Open reading frames are maintained by the flexible use of six-base pair blunt-end restriction sites that are not present in the DNA sequence of the plasmid cloning vector. Rapid production and analysis of plasmid intermediates are achieved by standard recombinant DNA cloning methods. The use of an anchored sticky-end restriction site and a variable blunt-end restriction site in each step of the cloning scheme gives specific orientation to each oligonucleotide fragment and results in high cloning efficiencies. The flexibility of the method allows for the construction of a gene of any size. Very large synthetic genes can be made by generating the intermediate gene fragments in parallel vectors and simply cloning the fragments in frame to produce the final construct.