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Nucleic Acids Research Advance Access first published online on August 31, 2006
This version published online on September 8, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl605
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© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Tapping diversity lost in transformations—in vitro amplification of ligation reactions

Daniel Christ*, Kristoffer Famm and Greg Winter

*To whom correspondence should be addressed. Tel: +44 1223 402106; Fax: +44 1223 412178; Email: duc{at}mrc-lmb.cam.ac.uk

Received May 5, 2006. Revised July 21, 2006. Accepted August 3, 2006.

Molecular evolution is a powerful means of engineering proteins. It usually requires the generation of a large recombinant DNA library of variants for cloning into a phage or plasmid vector, and the transformation of a host organism for expression and screening of the variant proteins. However, library size is often limited by the low yields of circular DNA and the poor transformation efficiencies of linear DNA. Here we have overcome this limitation by amplification of recombinant circular DNA molecules directly from ligation reactions. The amplification by bacteriophage Phi29 polymerase increased the number of transformants; thus from a nanogram-scale ligation of DNA fragments comprising two sub-libraries of variant antibody domains, we succeeded in amplifying a highly diverse and large combinatorial phage antibody library (>109 transformants in Escherichia coli and 105-fold more transformants than without amplification). From the amplified library, but not from the smaller un-amplified library, we could isolate several antibody fragments against a target antigen. It appears that amplification of ligations with Phi29 polymerase can help recover clones and molecular diversity otherwise lost in the transformation step. A further feature of the method is the option of using PCR-amplified vectors for ligations.


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