Silent Mutagenesis
The Silent Mutagenesis tool can help you introduces restriction sites without changing the corresponding protein sequence.
Select the amino acids (from one to three adjacent residues) with the mouse in one of the 'Sequence Display' windows (including the Oligo Editor)
and then select 'Silent Mutagenesis' from the Restriction Enzymes pull down menu or mouse-right-click pop up menu. A list of sequence variations, ordered by mismatch,
that do not alter the coding sequence is displayed. Below each sequence, the name of the Restriction Enzyme which has a site introduced (+) or deleted (-)
is shown together with the total number of sites for that enzyme in the complete DNA sequence. To introduce one of the suggested mutations into your main sequence, press the 'Use'
button at the top of the display. You can select the choice of enzymes considered (eg common6+ is useful) using the 'Enzyme Box Combo' button.
For example, in the sequence
CAGGCGCTCAGTGGGCTGCACCTGTCCGCAGACAGGCTG
Q A L S G L H L S A D R L
L H L defines part of a nuclear exclusion sequence. Select each L of LHL and mutate it to A, choosing any codon choice, then right click and select Silent Mutagenesis. DNADynamo produces a table showing you sequence variations and enzymes sites introduced or destroyed. Selecting the Enzyme Box common6+ usefully limits the results. The display shows you that using the codon GCG for both occurrences of A would introduce an Mlu1 site.
Note - comparison of enzyme site usage is with the original unmutated sequence (in the examples case CTGCACCTG) rather than the mutated AHA variant. In this way it is easy to design oligos intended to introduce mutations into a protein that also introduce restriction enzyme sites, enabling a rapid analysis of the products of the mutagenesis reaction. Other uses are also possible.