Diversity visualization by endonuclease: A rapid assay to monitor diverse nucleotide libraries

作者：Lim Theam Soon; Schuetze Tatjana; Lehrach Hans; Gloekler Joern; Konthur Zoltan^*

来源：Analytical Biochemistry, 2011, 411(1): 16-21.

DOI：10.1016/j.ab.2010.12.024

• S1 nuclease
• Diversity Assay
• Nucleotide library
• Phage display
• Mutagenesis

Many experiments require a fast and cost-effective method to monitor nucleic acid sequence diversity. Here we describe a method called diversity visualization by endonuclease (DiVE) that allows rapid visualization of sequence diversity of polymerase chain reaction (PCR) products based on DNA hybridization kinetics coupled with the activity of a single-strand specific nuclease. The assay involves only a limited number of steps and can be performed in less than 4 h, including the initial PCR. After PCR, the homoduplex double-stranded DNA (dsDNA) is denatured and reannealed under stringent conditions. During the reannealing process, incubation with Si nuclease removes single-stranded loops of formed heteroduplexes and the resulting digest is visualized on agarose gel. The sequence diversity is inversely proportional to the band intensities of Si nuclease surviving dsDNA molecules of expected size. As an example, we employed DiVE to monitor the diversity of panning rounds from a single-framework, semisynthetic single-chain antibody fragment (scFv) phage display library. The results are in good agreement with the observed decrease in diversity in phage display panning rounds toward the selection of monoclonal scFv. We conclude that the DiVE assay allows rapid and cost-effective monitoring of diversities of various nucleotide libraries and proves to be particularly suitable for scaffold-based randomized libraries.

• 出版日期2011-4-1