Shuffle optimizer: A program to optimize DNA shuffling for protein engineering

John N. Milligan, Daniel J. Garry

Research output: Chapter in Book/Report/Conference proceedingChapter

3 Scopus citations


DNA shuffling is a powerful tool to develop libraries of variants for protein engineering. Here, we present a protocol to use our freely available and easy-to-use computer program, Shuffle Optimizer. Shuffle Optimizer is written in the Python computer language and increases the nucleotide homology between two pieces of DNA desired to be shuffled together without changing the amino acid sequence. In addition we also include sections on optimal primer design for DNA shuffling and library construction, a smallvolume ultrasonicator method to create sheared DNA, and finally a method to reassemble the sheared fragments and recover and clone the library. The Shuffle Optimizer program and these protocols will be useful to anyone desiring to perform any of the nucleotide homology-dependent shuffling methods.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages11
StatePublished - Jan 1 2017

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745


  • Codon optimization
  • Computer program
  • DNA shuffling
  • Protein engineering
  • Protein library
  • Python
  • Random chimeragenesis on transient templates (RACHITT)
  • Staggered extension process (StEP)

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