Analyze Silent Mutations under the Cloning menu ﬁnds restriction sites that could be introduced by one or more synonymous mutations to a coding sequence.
To specify the set of restriction enzyme sites you want to introduce, ﬁrst create a new enzyme set containing the restriction enzymes you require. See section 14.3 for how to create a new enzyme set. Then open the Analyze Silent Mutations options and select this list in the Restriction Enzymes drop down.
To limit the number of substitutions required to generate a restriction site, check Limit maximum substitutions to and set it to the number you require.
Analyze Silent Mutations can be run on either the entire sequence, or a selected region. If no region is selected, Geneious will use the ﬁrst reading frame starting from base 1 of the sequence to determine the synonymous substitution sites. If you use a selected region of sequence, Geneious will use frame 1 of the selected region, unless you select an annotation with a diﬀerent frame speciﬁed in the annotation properties (e.g. a CDS annotation with the qualiﬁer codon_start=2 will use frame 2).
Once the analysis has run, green Potential Restriction Site annotations will be generated on the sequence. To see the modiﬁcation required to create a particular restriction site, mouse over the annotation. To apply the modiﬁcation to the sequence, ﬁrst click Allow Editing. Then click on the potential restriction site annotation to select it, right click and choose Apply potential restriction site from the drop down menu. The sequence will then be edited with the modiﬁcation required, and the potential restriction site annotation will be changed to a regular restriction site annotation. Figure 14.7 shows an example site before and after applying the change to create the new site.
Note that if an intact restriction site is present in the selected region then a blue Restriction Site annotation for the site will be added to the sequence.
To remove existing restriction sites from a sequence using synonymous substitutions, use Optimize Codons (see Section 14.13 ).