Stability in Solution: 2-3 days

Trimer Phosphoramidites

In principle, the simplest approach for oligonucleotide-directedmutagenesis would be the use of trimer phosphoramidites. Of the 64 possiblecombinations of codons, only 20 codons would be required to cover the 20 aminoacids, although, in practice, several codons will likely be duplicated dependingon the organism. Our trimers use the protection scheme described^1-3 by Kayushin et al. There is a concern that the sequence of the trimers has to beverified. For example, CAT coding for histidine, has to be differentiated fromTAC, coding for tyrosine. These two trimers have virtually identicallipophilicity and their identity cannot be clearly confirmed by HPLC. Thisproblem has been solved⁴ using HPLC electrospray mass spectrometricanalysis of the trimers, which provides data confirming molecular weight andsequence.

In Table 1, the trimers, their coding amino acid and theirreaction factor (RF) are listed. The reaction factor is critical since thetrimers will likely be mixed and they have differing reactivity in the couplingreaction. RF for AAC is 1.0 and for TAC is 1.6. Therefore, 1.6 equivalents ofTAC are needed for every 1.0 equivalent of AAC for equal coupling. Mixtures caneasily be made using equimolar solutions or the molecular weight of each trimerhas to be used to generate the appropriate weights of each trimer to use ifmixing by weight. An example of the preparation of a mixture of all 20 trimersis shown in the right column of Table 1 and completed in the footnotes.

All of the trimers are now available individually so thatresearchers can prepare custom mixtures. A mixture of all 20 trimers designed toproduce equal coupling of all 20 is also available. If you require customproduction of a specific mixture, please e-mail support@glenresearch.com for aquotation and projected delivery.

TABLE 1: TRIMER CODING AND PHYSICAL PARAMETERS