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Catalogue number: 103-20Description: white to off-white powderStorage of dry compound: 1 year at -20ºCProtein mutagenesis can be used to fine tune avariety of properties, such as improved stabilityto high temperatures, denaturants, or non-aqueoussolvents; higher affinity binding to atarget molecule; increased rates of enzymaticreactions; or changes of specificities. However,generating and finding these improved proteinscan be a difficult task. One of the mostpopular methods is to make pools of degenerateoligonucleotides, which can be incorporatedinto the genes as cassettes or by PCR by usingthe degenerate oligo as a primer.1 Degenerateoligonucleotides are synthesized as a mixture ofA/C/G/T phosphoramidites (N) at the site of thecodons to be mutated. Problems arise, though,from using an equimolar solution of each base.First there is a coding bias. Out of the 64 possiblecodon combinations of A, C, G and T, 18 codefor leucine, arginine or serine, but only 2 fortryptophan or methionine. As a result, only 3%of the mutagenic oligonucleotides will containmethionine or tryptophan, and over 28% willcontain either leucine, arginine or serine. Inaddition, the three nonsense codons will leadto chain termination in 4.7% of the sequences.There are ways to improve this situation. Forinstance, using two degenerate mixes of bases,N and G/C, on the DNA synthesizer to insertNNG/C into the sequence will halve the numberof the most degenerate codons, but still code forall 20 amino acids. However, still 59% of theclones will code for just eight amino acids and3% will have a stop codon inserted.The generation of redundant sequences and stopcodons makes searching a clonal library inefficient.However, it is possible to improve the efficiency ofthis process by using a mixture of trinucleotide (trimer)phosphoramidites.25 By synthesizing a set oftrimers that cover all 20 amino acids, the mutationof a gene can be carried out at the codon level ratherthan at individual bases. Therefore, unlike othermethods of mutagenesis, trimer phosphoramiditeslead to no codon bias, no frame-shift mutations,and no production of stop codons, making themone of the most efficient tools to explore sequencespace in protein regions that are important for function6 even in nonsaturating conditions.7, 8References: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, 1992.89,3581-3585.Price and Ordering Information
