‘Sticky feet’-directed mutagenesis and its application to swapping antibody domains

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Abstract
We describe a novel technique for precisely cutting and pasting two DNA seuqences without using restriction sites. The method is based on site-directed mutagenesis and uses a long primer, generated by the polymerase chain reaction (PCR), to transfer large segments of DNA into a single-stranded template. The primer anneals to the template by virtue of ''sticky feet'' sequences (complementary to the template) which are introduced at the ends of the primer by the PCR. Yields of desired recombinants were high (.apprx.36%) and the transplanted sequences (.apprx.400 bp) free of errors. We have used this technique to swap CH2 domains between two mouse antibodies, and find that this domain can carry features critical for triggering complement lysis, in addition to the C1q binding motif.