GeneArt Combinatorial DNA Libraries

What is a combinatorial DNA library?

In a combinatorial DNA library, multiple nucleotide positions can be mutated within the sequence and mutations at different positions can be combined with each other. Based on true rational design, combinatorial DNA libraries can achieve a maximum of diversity to screen for and identify synergistic beneficial mutations, e.g., affinity maturation via phage display libraries.

Take control using TRIM technology

Besides randomization via degenerate codons (e.g., NNS), the sequences of GeneArt Combinatorial DNA Libraries can also be diversified using preassembled trinucleotide building blocks (trinucleotide mutagenesis (TRIM) technology) within the chemical synthesis process, to achieve significantly higher quality than by using conventional technologies. This allows for complete customization of the amino acid composition at randomized sites and thus avoids the occurrence of unwanted stop codons or amino acids. Achieve an extra level of QC by requesting optional next-generation sequencing of your combinatorial DNA libraries using the Thermo Scientific Ion Torrent sequencing platform (see case study below).

Advantages of TRIM technology

  • Control every important feature of the library using TRIM technology—creates rational diversity where it is likely to have the most impact
  • Helps significantly reduce screening efforts compared to conventional mutagenesis methods, thus helping to save time and budget
  • Basically no design restrictions

GeneArt Combinatorial DNA Libraries

GeneArt Combinatorial DNA Libraries

Figure 1. Comparison of sequence randomization using degenerate codons (e.g. NNS, N: all 4 bases at 25%; S: G & C at 50% each) vs. TRIM technology.

Get Your Project Started

Please download the GeneArt Combinatorial Libraries Custom Service Requirements Form  to submit project information. For secure data transfer please register at our customer portal.

Libraries made with conventional technology by the incorporation of degenerate codons (NNS, VNS, etc.) are also available. Please use the above-mentioned questionnaire for both technologies.

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

Workflow for DNA library production including QC steps

Quality control:

Sequencing of backbone region (100% correctness)

Quality control:

RT-PCR

Quality control:

Bulk sequencing

Quality control:

Peer group sequencing, NGS (optional)

Step 1 : Oligosynthesis for generation of backbone and degenerate region(s)

Quality control:

Sequencing of backbone region (100% correctness)

Step 2 : Assembly of the nonamplified library

Quality control:

RT-PCR

Step 3 : Generation of amplified library

Quality control:

Bulk sequencing

Step 4 : Cloning into customer vector

Quality control:

Peer group sequencing, NGS (optional)

Step 5 : Shipment
  • Optimize antibody affinity (affinity maturation)
  • Humanize antibodies
  • Modify antibody specificity
  • Improve physicochemical protein properties such as solubility, heat stability, and activity in industrial environments
  • Enhance function, such as promoter activity and enzymatic properties
  • Prolong half-life of therapeutics for "in vivo" environments
  • Improve enzyme function for industrial use
  • Modify enantioselectivity of enzymes
  • Induce changes to alter proteins from patented sequences
GeneArt Ready-to-Clone Combinatorial Library
  • 2–5 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 Combinatorial 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

All GeneArt Combinatorial Library 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 amino acid content and distribution have been met, and to verify sequence integrity of unmutated portions of the construct
  • Bulk sequencing to verify that nondegenerate portions of the construct have the correct sequence and randomized portions meet customer’s specifications
  • 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)
  • Success rate: Customized design allows to include only substitutions known to contribute to a certain property which increases the likelihood of screening success
  • Flexibility: Even the ratio of allowed amino acids can be accurately controlled, for example, with ratios reflecting exactly the ones found in humans
  • Accuracy: TRIM significantly reduces frameshift mutations and "abolishes"  the occurrence of stop codons which makes the screening of e.g. antibody libraries more efficient. Ancillary mutation rates achieved are typically in a (correctness range of >82%)
  • Maximum diversity: Achievable library diversities of up to 1012
  • Speed: Our capacities allow for fast production times that are project-specific
Applications
  • Optimize antibody affinity (affinity maturation)
  • Humanize antibodies
  • Modify antibody specificity
  • Improve physicochemical protein properties such as solubility, heat stability, and activity in industrial environments
  • Enhance function, such as promoter activity and enzymatic properties
  • Prolong half-life of therapeutics for "in vivo" environments
  • Improve enzyme function for industrial use
  • Modify enantioselectivity of enzymes
  • Induce changes to alter proteins from patented sequences
Delivery formats
GeneArt Ready-to-Clone Combinatorial Library
  • 2–5 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 Combinatorial 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
Quality control

All GeneArt Combinatorial Library 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 amino acid content and distribution have been met, and to verify sequence integrity of unmutated portions of the construct
  • Bulk sequencing to verify that nondegenerate portions of the construct have the correct sequence and randomized portions meet customer’s specifications
  • 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)
Advantages of TRIM technology
  • Success rate: Customized design allows to include only substitutions known to contribute to a certain property which increases the likelihood of screening success
  • Flexibility: Even the ratio of allowed amino acids can be accurately controlled, for example, with ratios reflecting exactly the ones found in humans
  • Accuracy: TRIM significantly reduces frameshift mutations and "abolishes"  the occurrence of stop codons which makes the screening of e.g. antibody libraries more efficient. Ancillary mutation rates achieved are typically in a (correctness range of >82%)
  • Maximum diversity: Achievable library diversities of up to 1012
  • Speed: Our capacities allow for fast production times that are project-specific

Advantages of NGS quality control – a case study

  • Accurate: determination of library correctness, amino acid distribution, and uniqueness of clones
  • Comprehensive: delivers millions of reads for analysis
  • Reliable: sequence at clonal level with a proprietary technology and software solution

A GeneArt Combinatorial DNA Library from our production workflow was characterized using our current standard quality control protocol (“CE-sequenced”) and our novel next-generation sequencing (NGS)-based quality control for combinatorial DNA libraries (“NGS-sequenced”). The expected amino acid distribution is shown in the right-most panel (“expected”); in this case cysteines were not desired in the randomized positions. Our current standard QC protocol entails CE sequencing of at least 96 library clones and subsequent statistical analysis of the amino acid distribution at randomized positions. In this case, 304 variant clones were analyzed. In contrast, our novel NGS-based quality control for combinatorial DNA libraries on the Ion PGM (Personal Genome Machine) Sequencer allows for the analysis of tens of thousands of library clones. In this case, 41,625 clones were analyzed.

The table shows the percentage at which each amino acid appears in that position for 4 codons (X1 – X4). Note that in the CE-sequenced peer group the codons for two amino acids were not found, although they are present in the library, as shown by NGS sequencing (red and green circles).

The research leading to results regarding NGS quality control has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number 613931.

Table 1. Comparison of standard to NGS sequencing quality control for GeneArt Combinatorial DNA Libraries.

Comparison of standard to NGS sequencing quality control for GeneArt Combinatorial DNA Libraries
Options for DNA libraries
 GeneArt Strings DNA LibrariesGeneArt Combinatorial LibrariesGeneArt Combinatorial Libraries with Next-generation sequencing
AdvantageCost efficient; fastAs described aboveAs described above
Design flexibility+
Full IUPAC code available; however limited control over occurring amino acids		+++
TRIM technology allows for the accurate determination of occurring amino acids and ratios		+++
TRIM technology allows for the accurate determination of occurring amino acids and ratios
Correctness+
Gene synthesis process used, more unintended mutations occur		+++
Error free template enables best possible correctness of results		+++
Error free template enables best possible correctness of results
Complexity<1011<1011
<1012  optional		<1011
<1012  optional
Cost++++++
Production time+++
10–15 business days		+
4–6 weeks		+
4–6 weeks
 Learn moreSee aboveSee above

Resources

Literature

FAQs

For Research Use Only. Not for use in diagnostic procedures.

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