Learn about mutagenic oligos and mutagenesis libraries


In eArray, mutagenesis libraries are collections of DNA oligonucleotides (oligos) that can be used to create families of variant genes. These genes can then be used to produce families of variant proteins for functional studies. Traditional mutagenesis methods that use radiation or chemical agents can only produce random mutagenesis, and the reactions are difficult to control. Standard oligonucleotide-directed site-specific mutagenesis methods can produce well-defined sequence changes, but the creation of large numbers of mutants can be both time- and cost-prohibitive. In contrast, the mutagenesis oligo libraries that can be created in eArray give you precise, codon-level control over the variants that are produced. Use the oligo libraries together with the Agilent QuikChange Oligo Library Mutagenesis System reagent kit to quickly create large libraries of well-defined mutants that are ideally suited for applications such as scanning mutagenesis and mutagenesis that is targeted to many specific amino acids of interest.

Mutational regions

eArray designs mutagenic oligos to target sequences that you enter. Target sequences can be genes or other DNA sequences of interest. Within each target sequence, you define non-overlapping mutational regions to which to apply one or more mutational strategies. See the figure below for an example.


Figure 1 – Target sequence with mutational regions defined. You can apply one or more mutational strategies to each mutational region.

Mutational strategies

Mutational strategies create families of sequences that alter codons in the target sequence. You apply mutational strategies on the amino acid level. Click here for descriptions of the mutational strategies available in eArray.

When you set up a mutational strategy, you also select the specific type of protein expression host that you intend to use. This lets you customize the codon usage for the mutated positions in the generated mutagenic sequences to maximize the efficiency of expression. eArray supports E. coli, yeast, mammalian, and insect host types. By default, eArray uses the most commonly-used codons in the selected protein expression host for each mutated amino acid. Alternatively, you can customize the codon usage for individual amino acids.

Structure of mutagenic oligos

To the mutant sequences that are generated for a given mutational region, eArray adds 5' and 3' sequences that include PCR primer annealing sites. The annealing sites accommodate custom PCR primers that are used later in the mutagenesis protocol to amplify the mutagenic oligos that are generated for the given mutational region. eArray optimizes both the length and position of the PCR primer annealing sites based on several criteria. You can order the PCR primers that eArray designs for your library from an oligo manufacturing vendor.

For each mutational region, the result is a set of homologous mutagenic oligos that have identical 5' and 3' end sequences. In all cases, the sequence of a mutagenic oligo exactly matches the corresponding region of the target sequence, except at the location(s) of the requested change(s). The mutagenic oligo set created for a representative mutational region appears in Figure 2, below.


Figure 2 – Set of mutagenic oligos (oligo set) generated for one mutational region in a target DNA sequence. All oligos in the set are the same length, and are identical in sequence to the corresponding region of the target sequence, except for the necessary base changes to create the requested amino acid changes. The mutational regions in the oligos are flanked on both the 5' and 3' ends with sequences that contain PCR primer annealing sites. If space on the oligos and within the target sequence allows, eArray can optimize both the length and position of the PCR primer annealing sites for the set.

Specifications for the creation of mutagenesis libraries in eArray

Structure of mutagenesis libraries

eArray groups all of the oligos that are created from a given mutational region with any mutational strategy into an oligo set. Oligo sets are collections of homologous sequences that have the same 5' and 3' ends that serve as PCR primer annealing sites. A mutagenesis library can contain 1 to 20 oligo sets.

The figure below illustrates an example of a mutagenesis library structure.


Figure 3 – Structure of a mutagenesis library. A – Two target sequences. Two mutational regions have been defined for one target sequence, and one mutational region has been defined for another. This generates a total of three oligo sets. Each oligo set contains a separate oligo for each variant of the mutational region that is produced by the selected mutational strateg(ies). Note the PCR primer annealing sites on the 5' and 3' ends of the oligos. B – Mutagenesis library created from the two target sequences in panel A. The mutagenesis library contains all of the oligo sets that were generated from the target sequences. The PCR primers that eArray designs for the library need to be purchased through an oligo manufacturing vendor.

To create a mutagenesis library, see Ways to create mutagenesis libraries.

To learn how to navigate the Mutagenesis application type, see Learn to navigate the Mutagenesis application type.


Status of mutagenesis libraries – Available actions

Each mutagenesis library has a status that determines the actions that you can take on it. You can select one of these statuses when you create a library:

The table below shows the specific actions that you can take on mutagenesis libraries of each status. Click the links for more information on each action.




Search for library

View library

Edit library


Submit library to Agilent Manufacturing


Download library files


Request quote for library


Delete library


* Libraries with Submitted status have already been submitted to Agilent Manufacturing.

See also

Learn to navigate the Mutagenesis application type

Ways to create mutagenesis libraries