Designing Randomized DNA Sequences Free of Restriction Enzyme Recognition Sites

Biotechnol J. 2018 Jan;13(1):10.1002/biot.201700326. doi: 10.1002/biot.201700326. Epub 2017 Oct 16.


DNA libraries containing random "barcodes" complicate synthetic biology workflows that utilize restriction enzymes since restriction sites can appear inside some barcodes. By removing bases at particular sites in the barcodes, it is possible to create semi-random pools of barcodes that do not contain any restriction sites. The challenge is to remove as few bases as possible to maximize the number of sequences in the pool while ensuring all sequences are free of restriction sites. The authors present CutFree, a computational approach to create pools of random DNA barcodes that lack a pre-defined set of restriction sites. The resulting pools can be inexpensively produced en masse with standard DNA synthesis techniques. CutFree is experimentally validated by blocking digestion of pools of barcodes designed to frequently contain restriction sites. Using CutFree, a pool of 1.3 billion barcodes that are free from recognition sites for 182 commercially available restriction enzymes is designed. CutFree is available as a software package and an online tool (

Keywords: bioinformatics; genetic engineering; genomic engineering; next-generation sequencing; synthetic biology.

MeSH terms

  • Algorithms
  • Base Sequence / genetics*
  • Computational Biology / methods
  • DNA Barcoding, Taxonomic*
  • DNA Restriction Enzymes / chemistry
  • DNA Restriction Enzymes / genetics
  • Gene Library
  • High-Throughput Nucleotide Sequencing / methods*
  • Software


  • DNA Restriction Enzymes