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Generate codon balanced libraries for mutagenesis with trimer modifications

Avoid stop codons and limit amino acid redundancy with Trimer 19 or Trimer 20 codon modifications

Incorporating oligo codon trimers into oligo libraries results in balanced encoding of amino acids and eliminates unwanted stop codons. Such oligo libraries are useful for mutagenesis experiments to prepare proteins for screening for potential improvements in biological function.

Jun 29, 2016

Revised/updated Jul 19, 2017

Optimizing oligonucleotide libraries for mutagenesis

Oligonucleotide libraries are commonly used for mutagenesis of proteins that can be screened for potential improvements in biological function, such as enhanced expression levels, stability, solubility, enzymatic activity, or binding affinity. While you can create libraries of sequences by simply having random bases incorporated into synthetic oligonucleotides, this method introduces stop codons and a higher proportion of certain amino acids, which increases the time and resources required to screen the resulting libraries. Alternatively, using the 3 degenerate bases NNK will reduce, but not eliminate the number of possible stop codons and improves proportional codon biases, but will not eliminate, the number of possible stop codons and codon biases.

These issues can be avoided by incorporating codon trimers into your oligos. IDT trimer mixes do not contain stop codons, and trimer mixes provide a balanced mix of the amino acid codons, resulting in equal representation of each unique codon.

Trimer codon mix options for balanced amino acid coding

IDT provides 2 trimer codon mixes, Trimer 19 Mix and Trimer 20 Mix, that can be incorporated into IDT oligos.

Trimer 19 contains an equal ratio of the following 19 codons (excluding CYS):

  • AAA, AAC, ACT, ATC, ATG

  • CAG, CAT, CCG, CGT, CTG

  • GAA, GAC, GCT, GGT, GTT

  • TAC, TCT, TGG, TTC

Trimer 20 contains an equal ratio of the following 20 codons (including CYS):

  • AAA, AAC, ACT, ATC, ATG

  • CAG, CAT, CCG, CGT, CTG

  • GAA, GAC, GCT, GGT, GTT

  • TAC, TCT, TGC, TGG, TTC

To limit synthesis complexity and facilitate fast delivery, we restrict the total length of oligos with trimer insertions to 100 total bases (counting each trimer as 3 bases). The number of codon trimer insertions within an oligonucleotide is also limited to 15. However, we are able to consider other designs outside of these specifications as non-catalog requests. Just contact us at noncat@idtdna.com.

Create your own custom trimer codon mixes

We can also incorporate a custom trimer codon into oligos. This allows you to customize the ratios of trimers incorporated. To order a custom trimer codon mix, email noncat@idtdna.com with your name, organization, and sequence designs. Oligos containing Custom Trimer Mixes can be ordered as standard desalted oligos (recommended for oligos containing mixes), provided that other modifications included in the sequences do not require HPLC.

Quick Facts: Trimer 19 and 20

Availability: DNA

Location: 5', internal, and 3'

Scales: 100 nmol to large scale

Purification: Standard desalt or HPLC

Ordering (modification codes):

  • /iTriMix19/

  • /iTriMix20/

 

Please email noncat@idtdna.com for custom trimer mix requests.

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