Random Substitutions

In a controlled randomization library, random nucleotides within a defined region will be mutated at a defined average mutation rate. GeneArt™ Controlled Randomization technology allows you to accurately direct the effective frequency of random nucleotide substitutions. And unlike error-prone PCR, you decide exactly which part(s) of the gene are affected—randomize the entire open reading frame or confine the mutagenesis to specific regions. Randomized constructs are rigorously controlled to help ensure that the unchanged portions of the sequence are unaffected and that randomized portions contain the distribution of nucleotides you require to encode the amino acid composition you’re looking for.

  • Library diversity of up to 1011 variants
  • TRIM technology optional available
  • Next-generation sequencing using the Thermo Scientific Ion Torrent™ sequencing platform optional available

Get your project started

Please  download the questionnaire to submit project information. For secure data transfer please register at our
customer portal.

For further information regarding this service or the order process, please contact geneartsupport@thermofisher.com.

Expected distribution of mutations in an example sequence of 1000 bp

Synbio DE11
  • Diversify antigen-binding sites of antibodies
  • Improve physicochemical protein properties such as solubility, heat stability, and activity in industrial environments
  • Enhance function, such as promoter activity and enzymatic properties
  • Optimize antibody affinity (in vitro affinity maturation)
  • Humanize antibodies
  • Prolong half-life of therapeutics for in vivo environments
  • Improve enzymes for industrial use
  • Modify enantioselectivity of enzymes
  • Induce changes to alter proteins from patented sequences
GeneArt™ Ready-to-Clone Controlled Randomization Library
  • 5 µg of linear DNA ready to clone via 5' and 3' restriction sites
  • Non-amplified library, 17–170 fmol (1010–1011 specimen variant diversity) depending on library diversity
  • Amplification primers
GeneArt™ Cloned Controlled Randomization Library
  • 30 µg of plasmid DNA cloned into the vector of your choice
  • 12 aliquots of 0.5 mL glyercol stocks
  • Non-amplified library, 17–170 fmol (1010–1011 specimen variant diversity) depending on library diversity
  • Amplification primers
  • Library diversity of up to 1011 variants
  • Precise selection of the average mutation rate (number of changes per construct)
  • Random mutagenesis throughout construct, or limited to defined positions
  • Transition/transversion ratio of 1:2
  • Fewer silent mutations
  • Low ancillary mutation rate
  • Fast production times

All GeneArt™ Controlled Randomization products are sequenced and subjected to statistical analysis to help ensure that they meet the following quality benchmarks:

  • Sequencing of up to 96 individual transformants to verify that customer’s specifications regarding nucleotide content and amino acid encoding have been met, and that sequence integrity of unmutated portions of the construct meets minimum requirements
  • Bulk sequencing to verify that nondegenerated portions of the construct have the correct sequence and randomized portions meet customer’s specifications and maintain an open reading frame
  • Real-time PCR prior to amplification to verify that library diversity objectives have been met
  • Additional, optional next-generation sequencing quality control (additional fee applies)
Applications
  • Diversify antigen-binding sites of antibodies
  • Improve physicochemical protein properties such as solubility, heat stability, and activity in industrial environments
  • Enhance function, such as promoter activity and enzymatic properties
  • Optimize antibody affinity (in vitro affinity maturation)
  • Humanize antibodies
  • Prolong half-life of therapeutics for in vivo environments
  • Improve enzymes for industrial use
  • Modify enantioselectivity of enzymes
  • Induce changes to alter proteins from patented sequences
Delivery formats
GeneArt™ Ready-to-Clone Controlled Randomization Library
  • 5 µg of linear DNA ready to clone via 5' and 3' restriction sites
  • Non-amplified library, 17–170 fmol (1010–1011 specimen variant diversity) depending on library diversity
  • Amplification primers
GeneArt™ Cloned Controlled Randomization Library
  • 30 µg of plasmid DNA cloned into the vector of your choice
  • 12 aliquots of 0.5 mL glyercol stocks
  • Non-amplified library, 17–170 fmol (1010–1011 specimen variant diversity) depending on library diversity
  • Amplification primers
Advantages
  • Library diversity of up to 1011 variants
  • Precise selection of the average mutation rate (number of changes per construct)
  • Random mutagenesis throughout construct, or limited to defined positions
  • Transition/transversion ratio of 1:2
  • Fewer silent mutations
  • Low ancillary mutation rate
  • Fast production times
Quality control

All GeneArt™ Controlled Randomization products are sequenced and subjected to statistical analysis to help ensure that they meet the following quality benchmarks:

  • Sequencing of up to 96 individual transformants to verify that customer’s specifications regarding nucleotide content and amino acid encoding have been met, and that sequence integrity of unmutated portions of the construct meets minimum requirements
  • Bulk sequencing to verify that nondegenerated portions of the construct have the correct sequence and randomized portions meet customer’s specifications and maintain an open reading frame
  • Real-time PCR prior to amplification to verify that library diversity objectives have been met
  • Additional, optional next-generation sequencing quality control (additional fee applies)

Resources

FAQs

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