Incorporating Synthetic Oligonucleotides via Gene Reassembly (ISOR): A versatile tool for generating targeted libraries

  • Herman A
  • Tawfik D
  • 138


    Mendeley users who have this article in their library.
  • 77


    Citations of this article.


The directed evolution of proteins has benefited greatly from site-specific methods of diversification such as saturation mutagenesis. These techniques target diversity to a number of chosen positions that are usually non-contiguous in the protein's primary structure. However, the number of targeted positions can be large, thus leading to impractically large library size, wherein almost all library variants are inactive and the likelihood of selecting desirable properties is extremely small. We describe a versatile combinatorial method for the partial diversification of large sets of residues. Our library oligonucleotides comprise randomized codons that are flanked by wild-type sequences. Adding these oligonucleotides to an assembly PCR of wild-type gene fragments incorporates the randomized cassettes, at their target sites, into the reassembled gene. Varying the oligonucleotides concentration resulted in library variants that carry a different average number of mutated positions that comprise a random subset of the entire set of diversified codons. This method, dubbed Incorporating Synthetic Oligos via Gene Reassembly (ISOR), was used to create libraries of a cytosine-C5 methyltransferase wherein 45 individual positions were randomized. One library, containing an average of 5.6 mutated residues per gene, was selected, and mutants with wild-type-like activities isolated. We also created libraries of serum paraoxonase PON1 harboring insertions and deletions (indels) in various areas surrounding the active site. Screening these libraries yielded a range of mutants with altered substrate specificities and indicated that certain regions of this enzyme have a surprisingly high tolerance to indels.

Author-supplied keywords

  • DNA shuffling
  • Deletions
  • Directed evolution
  • Indels
  • Insertions
  • Methyltransferase
  • PON1
  • Rational design

Get free article suggestions today

Mendeley saves you time finding and organizing research

Sign up here
Already have an account ?Sign in

Find this document


  • Asael Herman

  • Dan S. Tawfik

Cite this document

Choose a citation style from the tabs below

Save time finding and organizing research with Mendeley

Sign up for free