Unbound MEDLINE

Directed evolution of nucleotide-based libraries using lambda exonuclease.

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.

Links

  • FREE Publisher Full Text
  • Authors

    Lim BN, Choong YS, Ismail A, Glökler J, Konthur Z, Lim TS

    Institution

    Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia.

    Source

    BioTechniques 53:6 2012 Dec pg 357-64

    MeSH

    Bacteriophage lambda
    DNA Polymerase I
    Directed Molecular Evolution
    Exonucleases
    Fluorescent Dyes
    Gene Library
    Models, Genetic
    Viral Proteins

    Pub Type(s)

    Journal Article
    Research Support, Non-U.S. Gov't

    Language

    eng

    PubMed ID

    23227986