Fig. 2: Easy and fast optimization of primers for mutational library cloning.

Automated design of mutagenic primers using the tailored software Kozane. A Workflow. The program selects surface exposed residues and generates all possible primers based on antigen sequence to mutate user selected residues. All primers subsequently go through a series of quality control steps based on melting temperature (Tm), hairpin, homodimer formation and GC clamp. Finally, quality-verified primers, with a minimal difference in Tm, are selected based on their GC content. B Surface selection of SARS-CoV-2 RBD residues. Automated analysis of a SARS-CoV-2 RBD structure generated all positions with >20% RSA. After excluding alanine, glycine, cysteine, and proline (black), 78 positions (green) remained as the selected alanine mutation library. C Individual filtering of primers. 33930 primers (blue) representing 435 possible primers for each of the 78 mutations were generated and filtered based on TM (yellow), hairpin (green), homodimer and GC clamp (red) as illustrated. The homodimer category was not included in the figure, as no primer failed this step. D Final primers are selected with minimal Tm difference. Kozane selected final primers with a Tm of 61 °C- except for amino acid position 383, where the closest primer had a Tm of 62 °C. All selected primers provided the desired mutations in a one condition mutagenesis protocol.