Modified base compositions at degenerate positions of a mutagenic oligonucleotide enhance randomness in site-saturation mutagenesis

Site-saturation mutagenesis, using degenerate oligonucleotide primers, is a frequently used method in introducing various mutations in a selected target codon, Oligonucleotides that are synthesized using equimolar concentrations of nucleoside phosphoramidites (dA, dC, dG, dT) in the positions to be saturated, result in a mutant population that is biased towards the original nucleotides. We found that this bias could be eliminated by modifying the concentrations of nucleoside phosphoramidites during the oligonucleotide synthesis, We synthesized eight degenerate oligonucleotides to saturate eight different codons, and sequenced a total of 344 mutagenized codons, In six of these eight oligonucleotides, we reduced to varying extents the concentrations Of those nucleotides in the target positions that would form base pairs with the template, From the data, we analyzed the effects of different base compositions in the oligonucleotides when mutagenizing different codons, the influence of the positions of mismatches, and the significance of different non-Watson-Crick base pairs, Based on these results, we suggest levels to which different phosphoramidites should be reduced when synthesizing oligonucleotides for site-saturation mutagenesis.