title: Diversity visualization by endonuclease: a rapid assay to monitor diverse nucleotide libraries.
abstract: 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 4h, 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 S1 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 S1 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.
authors.affiliation: Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics, D-14195 Berlin, Germany.
authors.forename: Theam Soon
authors.initials: TS
authors.lastname: Lim
authors.forename: Tatjana
authors.initials: T
authors.lastname: Schütze
authors.forename: Hans
authors.initials: H
authors.lastname: Lehrach
authors.forename: Jörn
authors.initials: J
authors.lastname: Glökler
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Konthur
mesh_terms: D001483:Base Sequence; D001681:Biological Assay / Q000379:methods*; D004720:Endonucleases / Q000378:metabolism*; D015723:Gene Library; D007135:Immunoglobulin Variable Region / Q000276:immunology; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000235:genetics*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013696:Temperature; D013997:Time Factors; D017186:Titrimetry
chemical_list: D007135:Immunoglobulin Variable Region; D009711:Nucleotides; D004720:Endonucleases