Glen Research offers a unique line of products known as trimer phosphoramidites. These are trinucleotide reagents that allow customers to effectively synthesize oligonucleotides based on amino acid codons rather than individual nucleotides.1 For researchers looking to generate oligonucleotide libraries for mutagenesis, these reagents avoid stop codons and amino acid redundancy. In theory, such libraries can be obtained via a split and pool synthesis workflow without trimer phosphoramidites, but this is very awkward to perform in practice for a large number of codons or longer codon containing regions. The trimer phosphoramidite approach to mutagenesis library construction is distinguished from other methods by the ability to fully control total or partial randomization at each codon position of a gene, maximizing the efficiency of high throughput screening processes.
All of our trimers are available individually so that researchers can prepare custom trimer mixes. Two pre-made catalog trimer mixes are available: 13-1991-xx, for incorporating all 20 amino acid codons equally into a sequence and 13-1992-xx, for incorporating 19 amino acid codons (-Cys). For a custom trimer mix of a particular subset of codons or a trimer mix that represents a set of trimers that is biased toward a particular codon or codons, please contact [email protected] for a quotation and projected delivery date.
The reaction factor is critical since the trimers will likely be mixed and they have differing reactivity in the coupling reaction. In Table 1, the trimers, their coding amino acid and their reaction factor (RF) are listed. RF for AAC is 1.0 and for TAC is 1.6. Therefore, 1.6 equivalents of TAC are needed for every 1.0 equivalent of AAC for equal coupling. Mixtures can easily be made using equimolar solutions or the molecular weight of each trimer has to be used to generate the appropriate weights of each trimer to use if mixing by weight. An example of the preparation of a mixture of all 20 trimers is shown in the right column of Table 1 and completed in the footnotes.
Table 1: Trimer Coding and Physical Parameters
| Trimer | Amino Acid | Abbreviation | Molecular Weight (MW) | Reaction Factor (RF) | MW x RF | mg/10µmol (adjusted for RF) |
| AAA | Lys | K | 1911.5 | 1.1 | 2102.65 | 21.0 (11) |
| AAC | Asn | N | 1887.5 | 1.0 | 1887.50 | 18.9 (10) |
| ACT | Thr | T | 1774.5 | 1.3 | 2306.85 | 23.1 (13) |
| ATC | Ile | I | 1774.5 | 1.2 | 2129.40 | 21.3 (12) |
| ATG | Met | M | 1780.5 | 1.3 | 2314.65 | 23.1 (13) |
| CAG | Gln | Q | 1869.5 | 2.0 | 3739.00 | 37.4 (20) |
| CAT | His | H | 1774.5 | 1.9 | 3371.55 | 33.7 (19) |
| CCG | Pro | P | 1845.5 | 1.8 | 3321.90 | 33.2 (18) |
| CGT | Arg | R | 1756.5 | 1.1 | 1932.15 | 19.3 (11) |
| CTG | Leu | L | 1756.5 | 1.2 | 2107.80 | 21.1 (12) |
| GAA | Glu | E | 1893.5 | 1.9 | 3597.65 | 36.0 (19) |
| GAC | Asp | D | 1869.5 | 1.3 | 2430.35 | 24.3 (13) |
| GCT | Ala | A | 1756.5 | 1.5 | 2634.75 | 26.3 (15) |
| GGT | Gly | G | 1762.5 | 1.1 | 1938.75 | 19.4 (11) |
| GTT | Val | V | 1667.5 | 1.9 | 3168.25 | 31.7 (19) |
| TAC | Tyr | Y | 1774.5 | 1.6 | 2839.20 | 28.4 (16) |
| TCT | Ser | S | 1661.4 | 1.3 | 2159.82 | 21.6 (13) |
| TGC | Cys | C | 1756.5 | 1.5 | 2634.75 | 26.3 (15) |
| TGG | Trp | W | 1762.5 | 2.4 | 4230.00 | 42.3 (24) |
| TTC | Phe | F | 1661.4 | 1.3 | 2159.82 | 21.6 (13) |
| =530.0 mg |
Prepare 592.6 mg of the trimer mix, taking the amount (mg) for each trimer from the right column. Dissolve the trimer mix in dichloromethane (highest grade possible; acid-free). Evaporate to dryness to produce a homogenous mixture of all 20 trimers.
Dissolve 592.6 mg, which is equivalent to 20X10 µmoles (normalized for RF) of the trimer mix in 2.0 mL of acetonitrile-dichloromethane mixture, 1:3 v/v to produce a 0.10N solution of trimers, ready for use in a synthesizer.
References
1. A. Yagodkin, et al., Nucleosides, Nucleotides and Nucleic Acids, 2007, 26, 473 - 497.