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          Institute: MPI für molekulare Genetik     Collection: Department of Vertebrate Genomics     Display Documents



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ID: 556507.0, MPI für molekulare Genetik / Department of Vertebrate Genomics
Diversity visualization by endonuclease: A rapid assay to monitor diverse nucleotide libraries.
Authors:Lim, T. S.; Schutze, T.; Lehrach, H.; Glökler, J.; Konthur, Z.
Language:English
Date of Publication (YYYY-MM-DD):2011-04-01
Title of Journal:Analytical Biochemestry
Journal Abbrev.:Anal Biochem
Volume:411
Issue / Number:1
Start Page:16
End Page:21
Copyright:© 2010 Elsevier Inc. All rights reserved.
Review Status:not specified
Audience:Experts Only
Abstract / Description: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.
Free Keywords:S1 nuclease;
Diversity Assay;
Nucleotide library;
Phage display;
Mutagenesis
Comment of the Author/Creator:Email:konthur@molgen.mpg.de
External Publication Status:published
Document Type:Article
Communicated by:Hans Lehrach
Affiliations:MPI für molekulare Genetik
External Affiliations:1.Department of Biology, Chemistry, and Pharmacy, Free University Berlin, D-14195 Berlin, Germany;
2.Institute for Research in Molecular Medicine, University Science Malaysia, 11800 Penang, Malaysia.
Identifiers:ISSN:0003-2697 [ID No:1]
URL:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=... [ID No:2]
DOI:10.1016/j.ab.2010.12.024 [ID No:3]
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