Directed evolution of nucleotide-based libraries using lambda exonuclease

作者：Lim Bee Nar; Choong Yee Siew; Ismail Asma; Gloekler Joern; Konthur Zoltan; Lim Theam Soon^*

来源：Biotechniques, 2012, 53(6): 357-364.

DOI：10.1144/000113964

• Lambda Exonuclease
• Antibody
• Mutagenesis
• Diversity
• Nucleotide-based Libraries

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 2x10(8) and 6x10(7), respectively. We conclude that our method is an efficient and convenient approach to generate diversity in nucleic acid based libraries, especially recombinant antibody libraries.

• 出版日期2012-12