Synthesis of mutant parathyroid hormone genes via site-specific recombination directed by crossover linkers

A synthetic ‘crossover linker’ technique has been designed for gene modification. The linker has (i) a restriction end for an initial ‘cohesive end’ ligation with one terminus of a linearized plasmid, (ii) a middle section carrying modified sequence information, and (iii) an ‘homology-searching’ sequence of 20 bp at its other end, that is homologous to a specific region in the opposite terminus of the plasmid. Inside the Escherichia coli transformation host, intramolecular recombination between the homologous ends of the resultant plasmid intermediate completes the integration of the Unker. Using different crossover Unkers, a human parathyroid hormone gene which had previously been cloned into plasmid pUC8 was converted to mutant coding sequences via specific (i) base substitution, (ii) sequence deletion and (iii) sequence insertion.