'Sticky feet'-directed mutagenesis and its application to swapping antibody domains.
We describe a novel technique for precisely cutting and pasting two DNA sequences 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 (approximately 36%) and the transplanted sequences (approximately 400bp) 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.
Clackson
T
T
MRC Laboratory of Molecular Biology, Cambridge, UK.
Winter
G
G
D000818:Animals; D001483:Base Sequence; D003165:Complement System Proteins / Q000276:immunology; D004247:DNA / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000235:genetics; D005091:Exons; D006461:Hemolysis; D007074:Immunoglobulin G / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins; D019914:Taq Polymerase; D013698:Templates, Genetic; D014162:Transfection
D007074:Immunoglobulin G; D011994:Recombinant Proteins; D003165:Complement System Proteins; D004247:DNA; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase