Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia.
Biotechniques. 2012 Dec;53(6):357-64. doi: 10.2144/000113964.
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.
利用重组或诱变定向进化核苷酸文库是定制蛋白质催化或生物物理特性的重要技术。然而，传统的定向进化方法存在繁琐的消化和连接步骤。在这里，我们描述了一种使用单链 DNA (ssDNA) 作为起始模板来增加核苷酸多样性的简单方法。使用带有重叠区域的磷酸化引物进行初始 PCR 扩增，然后用 lambda 核酸外切酶处理，生成可以通过重叠区域退火的 ssDNA 模板。然后通过 Klenow 片段延伸生成双链 DNA (dsDNA)。为了证明该方法在核苷酸文库定向进化中的适用性，我们分别生成了基因重排和区域诱变的合成抗体文库，滴度分别为 2×108 和 6×107。我们得出结论，我们的方法是一种在基于核酸的文库中产生多样性的有效且方便的方法，特别是重组抗体文库。
