Limitations of Conventional Codon Randomization with Oligonucleotide Synthesis
- No flexibility in choosing codons for protein expression optimization.
- The NNN library has six fold difference in amino acid representation; the amount of the expressed full-length sequences drops quickly due to stop codon termination and codon-dependent low expression, and thus
NNN is not suitable for mutations of more than ten residues.
- The NNC/T library is incomplete in representing amino acids (missing five of the 20 amino acids).
- The NNG/T and NNC/G/T libraries overcomes some limitations but still suffer from the difficulties of being inflexible in codon choices and sequence bias.
- The codon-based trinucleotide synthesis for building oligonucleotides provides specific codon for each amino acid, but it is expensive to be of routine use.
- None of the above method would allow deletion or insertions.
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