Efficient recombination of Lym-1 scFv gene using multiple doubly-restricted DNA fragments

dIn order to improve radioimmunotherapy of lymphoma, a Lym-1 single-chain a ntigen-binding (scFv) protein molecule was produced. Because the commonly u sed polymerase chain reaction (PCR) method frequently causes unexpected mut ations, we developed a non-PCR method for scFv gene assembly. The method in volved a stepwise linkage of doubly-restricted DNA fragments and re-digesti on of the resultant concatamers. Using this strategy, the Lym-1 scFv expres sion gene was readily constructed without mutations. The recombinant gene w as cloned into an expression vector and scFv protein was expressed. The met hod can be used for other genes or DNA recombination.