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  Directed evolution of nucleotide-based libraries using lambda exonuclease

Lim, B. N., Choong, Y. S., Ismail, A., Glökler, J., Konthur, Z., & Lim, T. S. (2012). Directed evolution of nucleotide-based libraries using lambda exonuclease. Biotechniques, 53(6), 357-364. doi:10.2144/000113964.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-000E-F06E-8 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-000E-F06F-6
Genre: Journal Article

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© 2012 BioTechniques
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 Creators:
Lim, B. N., Author
Choong, Y. S., Author
Ismail, A., Author
Glökler, J.1, Author              
Konthur, Z.2, Author              
Lim, T. S., Author
Affiliations:
1Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society, Berlin, Germany, ou_1433550              
2In vitro Ligand Screening (Zoltán Konthur), Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society, Berlin, Germany, ou_1479653              

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 Abstract: 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 2x108 and 6x107, respectively. We conclude that our method is an efficient and convenient approach to generate diversity in nucleic acid based libraries, especially recombinant antibody libraries.

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Language(s): eng - English
 Dates: 2012-12
 Publication Status: Published in print
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 Rev. Method: Peer
 Identifiers: DOI: 10.2144/000113964
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Title: Biotechniques
  Other : Biotechniques
Source Genre: Journal
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Publ. Info: [Natick, MA : Eaton Pub. Co.
Pages: - Volume / Issue: 53 (6) Sequence Number: - Start / End Page: 357 - 364 Identifier: ISSN: 0736-6205
CoNE: /journals/resource/954925536105