Directed evolution of nucleotide-based libraries using lambda exonuclease
TLDR
A simple method to increase nucleotide diversity using single-stranded DNA (ssDNA) as a starting template and an efficient and convenient approach to generate diversity in nucleic acid based libraries, especially recombinant antibody libraries is described.Abstract:
Directed evolution of nucleotide libraries using recombination or mutagenesis is an important technique for customizing catalytic or biophysical traits of proteins. Conventional directed evolution methods, however, suffer from cumbersome digestion and ligation steps. Here, we describe a simple method to increase nucleotide diversity using single-stranded DNA (ssDNA) as a starting template. An initial PCR amplification using phosphorylated primers with overlapping regions followed by treatment with lambda exonuclease generates ssDNA templates that can then be annealed via the overlap regions. Double-stranded DNA (dsDNA) is then generated through extension with Klenow fragment. To demonstrate the applicability of this methodology for directed evolution of nucleotide libraries, we generated both gene shuffled and regional mutagenesis synthetic antibody libraries with titers of 2×108 and 6×107, respectively. We conclude that our method is an efficient and convenient approach to generate diversity in nucleic acid based libraries, especially recombinant antibody libraries.read more
Citations
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Polishing the craft of genetic diversity creation in directed evolution
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Phage display antibodies for diagnostic applications.
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TL;DR: The generation of monospecific binders provides a vital tool for diagnostics at a lower cost and higher efficiency and the flexibility to modify recombinant antibodies allows great applicability to various platforms for use.
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Cognizance of Molecular Methods for the Generation of Mutagenic Phage Display Antibody Libraries for Affinity Maturation.
TL;DR: A review of phage display technology used for antibody generation can be found in this paper, where the authors provide a brief account of PHV technology and a summary of different combinatorial library characteristics.
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Principles and application of antibody libraries for infectious diseases
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References
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TL;DR: It is reported here that selected mutants had a minimum inhibitory concentration of 640 μg ml-1, a 32,000-fold increase and 64-fold greater than any published TEM-1 derived enzyme.
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Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis
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Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity
TL;DR: Single-chain antibody mutants have been evolved in vitro with antigen-binding equilibrium dissociation constant K(d) = 48 fM and slower dissociation kinetics than those for the streptavidin-biotin complex, demonstrating that the antibody Fv architecture is not intrinsically responsible for an antigen- binding affinity ceiling during in vivo affinity maturation.
Journal ArticleDOI
Molecular evolution by staggered extension process (StEP) in vitro recombination
TL;DR: The staggered extension process (StEP) consists of priming the template sequence(s) followed by repeated cycles of denaturation and extremely abbreviated annealing/polymerase-catalyzed extension, which results in recombination of polynu-cleotide sequences.