Trinucleotide (TRIMER CODON) Phosphoramidites
Catalogue number: 103-20
Description: white to off-white powder
Storage of dry compound: 1 year at -20ºC
Protein mutagenesis can be used to fine tune a
variety of properties, such as improved stability
to high temperatures, denaturants, or non-
solvents; higher affinity binding to a
target molecule; increased rates of enzymatic
reactions; or changes of specificities. However,
generating and finding these improved proteins
can be a difficult task. One of the most
popular methods is to make pools of degenerate
oligonucleotides, which can be incorporated
into the genes as cassettes or by PCR by using
the degenerate oligo as a primer.1 Degenerate
oligonucleotides are synthesized as a mixture of
A/C/G/T phosphoramidites (N) at the site of the
codons to be mutated. Problems arise, though,
from using an equimolar solution of each base.
First there is a coding bias. Out of the 64 possible
codon combinations of A, C, G and T, 18 code
for leucine, arginine or serine, but only 2 for
tryptophan or methionine. As a result, only 3%
of the mutagenic oligonucleotides will contain
methionine or tryptophan, and over 28% will
contain either leucine, arginine or serine. In
addition, the three nonsense codons will lead
to chain termination in 4.7% of the sequences.
There are ways to improve this situation. For
instance, using two degenerate mixes of bases,
N and G/C, on the DNA synthesizer to insert
NNG/C into the sequence will halve the number
of the most degenerate codons, but still code for
all 20 amino acids. However, still 59% of the
clones will code for just eight amino acids and
3% will have a stop codon inserted.
The generation of redundant sequences and stop
codons makes searching a clonal library inefficient.
However, it is possible to improve the efficiency of
this process by using a mixture of trinucleotide (trimer)
phosphoramidites.2–5 By synthesizing a set of
trimers that cover all 20 amino acids, the mutation
of a gene can be carried out at the codon level rather
than at individual bases. Therefore, unlike other
methods of mutagenesis, trimer phosphoramidites
lead to no codon bias, no frame-shift mutations,
and no production of stop codons, making them
one of the most efficient tools to explore sequence
space in protein regions that are important for function
6 – even in nonsaturating conditions.7, 8
1. Zon, G., Gallo, K., Samson, C., Shao, K., Michael F. Summers,
M., Byrd, R. Nucleic Acids Res, 1985, 13, 8181-8196.
2. Kayushin, A., Korosteleva, M., Miroshnikov. Nucleos. Nucleot.
Nucleic Acids, 2000, 19, 1967-1976.
3. Kayushin, A., M. Korosteleva, . Miroshnikov, A. Nucleos Nucleot,
1999, 18, 1531-1533.
4. Kayushin, A., M. Korosteleva, . Miroshnikov, A. W. Kosch,
W., Zubov, D., Piel N. Nucleic Acids Res., 1996, 24, 3748-3755.
5. Mauriala, T., Auriola, S., Azhayev, A., Kayushin, A., Korosteleva,
M., Miroshnikov, A. J Pharm Biomed Anal, 2004. 34, 199-206.
6. Yagodkin, A., Azhayev, A., Roivainen, J., Antopolsky, M., Kayushin,
A., Korosteleva, M., Miroshnikov, A., Randolph, J., Mackie,
H. Nucleos. Nucleot. Nucleic Acids 2007, 26, 473-497.
7. Neylon, C. Nucleic Acids Res, 2004. 32, 1448-59.
8. Sondek, J. and D. Shortle, Proc Natl. Acad. Sci. U S A, 1992.
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