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Mutation Library Construction

VectorBuilder offers extensive expertise in constructing custom mutagenesis libraries. These libraries are critical for many studies as the initial step for probing the key residues and domains in proteins. We provide versatile strategies for creating your diverse pool of mutations, including chip-based synthesis, error-prone PCR, degenerate codon utilization, and DNA shuffling. These mutations can be confined to a single region or distributed across multiple regions along the sequence.


  • Diverse cloning strategies: Tailored to your research goals, we employ various approaches to construct your mutation library.
  • High complexity: We can generate mutation libraries exceeding 108 in complexity.
  • Great coverage and high uniformity: Our libraries typically achieve >98% coverage of targeted variants.

Service Details

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Price and turnaround Price Match
Service Module Brief Description Price (USD) Turnaround
Mutation library design VectorBuilder's team of highly experienced scientists will design your customized mutation library using versatile strategies. You have the option to select from our extensive collection of validated vector backbones. Free 1-4 days
Pooled library cloning Includes oligo/gene synthesis, massive parallel cloning of inserts for variable regions into desired vector backbone, preliminary validation by Sanger sequencing, and full validation by NGS. Deliverable includes library in E. coli glycerol stock and NGS report. From $2,300 5-8 weeks
Virus packaging of pooled library Please click here to view detailed info of virus packaging services. The price for packaging pooled library is 1.5-fold of the price for packaging single vector.
NGS deconvolution of post-screening sample Includes NGS library preparation from genomic DNA of screened cells, Illumina sequencing (>500x coverage), and data analysis. From $320 per sample 3-5 weeks

Technical Information

Mutation library construction strategiesMutation library construction strategies

Chip-based oligo synthesis

Chip-based oligo synthesis is ideal for generating pre-designed sequence variants with various sizes and sequences, such as saturation mutations for any given site. Our chip-based DNA synthesis can usually go up to 300 nt with a low error rate.

Error-prone PCR

Error-prone PCR is the most straightforward approach for creating highly variable mutation libraries which involves modifying standard PCR methods to mutagenize the original sequence. More specifically, error-prone PCR employs a combination of various sub-optimal PCR conditions including low fidelity polymerases, longer extension times, higher Mg2+ concentrations, the addition of Mn2+, and varying dNTP concentrations for introducing random point mutations into the original sequence.

Degenerate codon

The degeneracy of genetic code allows one amino acid to be encoded by multiple codons. Degenerate codons in oligonucleotide synthesis introduce controlled, highly randomized mutations. This approach enables targeted focus on regions and amino acids that are likely to yield positive hits. The NNK library, for example, a widely used degenerate mutation library, involves NNK degenerate primers encompassing 32 codon combinations (N= A/C/G/T, K= G/T), which encode all 20 amino acids and one stop codon. Utilizing degenerate codons in library construction is an effective way to introduce diversity.

Degenerate Codon # Codons Stop Codon Amino Acids
NNN 64 3 All 20
NNK / NNS 32 1 All 20

DNA shuffling

DNA shuffling is a highly efficient method to generate chimeric DNA sequences through homologous recombination. This technique involves the digestion of parental gene(s) into random fragments, followed by their reassembly into new variants using primer-less PCR, which relies on partial sequence homology for recombination. Alternatively, synthetic shuffling can be employed for creating high-complexity libraries, combining rational design with directed evolution. DNA family shuffling may also be employed following other mutagenesis methods such as error-prone PCR.

Experimental data
Nucleotide composition of (NNK)11 in the plasmid library

Figure 1. Nucleotide composition of an (NNK)11 plasmid library. The library was constructed using the NNK degenerated codon strategy. Each bar represents a distinct nucleotide position along the DNA sequence, with colors indicating the observed nucleotides. All positions exhibited anticipated nucleotides with observed frequencies close to theoretical values (N = 25% A + 25% T + 25% G + 25% C, K = 50% T + 50% G).

Distribution of amino acids in a 7-mer variable region of a plasmid library

Figure 2. Distribution of amino acids in a 7-mer variable region of a plasmid library. The library was constructed using the NNK degenerate codon strategy. Each bar represents the theoretical (teal) or actual (pink) percentage of specific amino acids or the stop codon. The observed frequencies of all 20 amino acids and the stop codon closely matched the theoretical frequencies.

mutationComplexity, coverage, and alignment rate of chip-based oligo libraries constructed by VectorBuilder.

Figure 3. Complexity, coverage, and alignment rate of chip-based oligo libraries constructed by VectorBuilder. (A) The library complexity ranged from 102 to exceeding 105. (B) Both the coverage and read alignment rate were very high, highlighting the exceptional coverage and low error rate of our libraries.

How to Order

Customer-supplied library plasmid pool

If the customer-supplied premade plasmid pools are used, please send us the materials following the Materials Submission Guidelines. Please strictly follow our guidelines to set up shipment to avoid any delay or damage of materials. All customer-supplied materials undergo mandatory QC by VectorBuilder which may incur a $100 surcharge for each item. Please note that production may not be initiated until customer-supplied materials pass QC. For customer-supplied premade plasmid pools, we cannot provide any guarantees regarding the complexity or uniformity of the library.


What are the advantages and limitations of different mutagenesis strategies for mutation library?
Strategy Advantages Limitations
Error-prone PCR
  • Rapid generation of mutants
  • Knowledge of the targeted sequence not required
  • Cost-effective
  • Higher chance of introducing multiple or unintended mutations, which poses challenges for precise control over the mutations
  • Challenging for mutation identification throughout the GOI by NGS
Degenerate codon
  • Generates a highly diverse pool of variants
  • Allows for incorporation of multiple different amino acids at certain locations
  • Avoids bias with proper degenerate codon selection
  • Possible of generating stop codon
  • Technically challenging and expensive for oligo synthesis
DNA shuffling
  • Higher chance of generating functional mutants
  • Can be combined with other mutagenesis approaches to increase variant diversity
  • Enables recombination of entire gene sequence
  • Possible of introducing unintended sequence combinations
  • Labor intensive
  • Challenging for full-length mutation identification by NGS
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