Tapping diversity lost in transformations--in vitro amplification of ligation reactions.
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 (>10(9) transformants in Escherichia coli and 10(5)-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.
Christ
Daniel
D
duc@mrc-lmb.cam.ac.uk
Famm
Kristoffer
K
Winter
Greg
G
D017103:Bacillus Phages / Q000201:enzymology*; D022801:Complementarity Determining Regions / Q000235:genetics; D004270:DNA, Circular / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase; D014644:Genetic Variation; D019151:Peptide Library; D016133:Polymerase Chain Reaction / Q000379:methods*; D013698:Templates, Genetic; D014170:Transformation, Genetic
D022801:Complementarity Determining Regions; D004270:DNA, Circular; D019151:Peptide Library; D004259:DNA-Directed DNA Polymerase