‘Sticky feet’-directed mutagenesis and its application to swapping antibody domains (original) (raw)
Journal Article
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MRC Laboratory of Molecular Biology
Hills Road, Cambridge CB2 2QH, UK
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MRC Laboratory of Molecular Biology
Hills Road, Cambridge CB2 2QH, UK
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Received:
01 November 1989
Revision received:
15 November 1989
Accepted:
15 November 1989
Published:
25 December 1989
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Tim Clackson, Greg Winter, ‘Sticky feet’-directed mutagenesis and its application to swapping antibody domains, Nucleic Acids Research, Volume 17, Issue 24, 25 December 1989, Pages 10163–10170, https://doi.org/10.1093/nar/17.24.10163
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Abstract
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 (∼36%) and the transplanted sequences (∼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.
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