Random mutagenesis is a powerful tool in protein structure-function analysis.
One approach is the synthesis of cDNA sequences using doped nucleoside phosphoramidites. Sequences can also be imported from and exported to GCG sequence file format. In addition to the insertion, deletion, and substitution of bases in the sequence, several features and ranges within the sequence can be defined. As the cloning of the synthetic cDNA pool is dependent on unique restriction enzyme sites encoded by the 5' and 3' PCR primers, any cDNAs with internal copies of these same sites will be fragmented upon restriction enzyme digestion and, hence, rendered unclonable. This anti-termination strategy therefore entails a set of three additional nucleotide mixes, one each for adenine, cytosine, and guanine in the first position of xAA, xAG, and xGA codons.