Silent Mutator is an online software application designed to generate restriction sites through silent mutagenesis. It scans a user-provided cDNA sequence for all potential new restriction sites whose introduction would not change the encoded protein sequence. It uses a highly efficient original algorithm that allows to quickly process even relatively long sequences (several kilobases in a few seconds) so unlike other silent mutation scanning programs Silent Mutator has no length limit for the input sequence. Moreover, it can scan for new sites of all commercially available restriction enzymes and can use any genetic code to translate and preserve a protein sequence.
Before starting any analysis you have to set a few parameters to match the input sequence character and your intentions. Set if the sequence is linear or circular. Choose which restriction sites should the app scan the sequence for. The two basic options are a "pre-set list of most commonly used enzymes" and "all commercially available enzymes". Both can be further modified with additional options. For the first option, clicking "edit the list" will display a restriction enzyme selection dialog where you can change the default list either manually or by providing a list of restriction enzymes in a text file. To change the selection manually, use the checkboxes located in each restriction enzyme row and click the Apply button. You can save the modified selection into a file for future use with the Save List button. To load your own list, click the Load List button and choose the file in the following dialog. The file must be a plain text file with one restriction enzyme name in each line. To restore the default selection, click the Reset button. To store the present selection for the rest of the current session, click the Set Permanent button. This function, however, may not work in some browsers.
The second option - all commercially available enzymes - is by default narrowed to restriction enzymes whose recognition site is at least 6 nt long and does not contain ambiguous positions. You can remove these limitations using the checkboxes located below the "all commercially available enzymes" option, but be aware that the high-frequency short recognition sites will heavily clutter the scanning results.
In the next step choose if the program should hide or display any Dam/Dcm-methylated sites. Finally, select the genetic code to be used in translation of the input sequence from the drop-down menu labeled "Genetic code". If you need to use a genetic code not offered in the drop-down menu, you can modify one of the selectable genetic codes by first selecting a genetic code from the menu and then changing the selection to "User Provided". The genetic code table can be completely replaced, but the format must stay exactly the same. You can find and copy-paste genetic codes from the Genetic Codes NCBI webpage
Silent mutation scanning
The sequence to be scanned for potential new restriction sites can be either directly pasted in the sequence text area or provided as a text file. In the latter case, use the Choose File button and subsequent dialog. The sequence file must be a plain text file (i.e. simple text without any formatting, no MS Word document!). The program can parse fasta files (sequences with a header in the form ">sequence name") and GenBank files (the database records that contain a nucleotide sequence plus related information and feature definitions). You can provide just the coding part of the cDNA sequence, but also a whole mRNA or an entire plasmid sequence. The last case is particularly useful if you aim to introduce sites that will be unique in the context of the whole plasmid. To start the scanning, select the sequence range of interest and click the Translate & Analyze button. If you provided the sequence as a GenBank file with the protein-coding region annotated as a feature, you can select the region simply by clicking on its name in the parsed cDNA annotation listing that appears below the "Choose File" button.
Understanding the results
After the scanning is complete, an annotated sequence will appear below the main program panel. Restriction sites already present are annotated in the first line just above the nucleotide sequence with unique sites in red. One-letter amino acid symbols located in the second line above the nucleotides indicate the protein translation of the chosen cDNA. All potential new restriction sites are listed from the third line above the nucleotides up. The positions of the labels are aligned with the first nucleotide position of the corresponding recognition site and the pink numbers indicate cutting positions. Moving the mouse cursor over a potential new restriction site label displays an alignment of the original sequence and the modified sequence with the new restriction site highlighted with red color.
In addition to the annotated sequence, a table of all existing and potential new restriction sites is generated after the scanning. This table is located either right to the main program panel or below the annotated sequence, depending on the width of the browser window.
Sequence modification and export
To modify the sequence so that a new restriction site is introduced, first find the name of the site in the table of existing and potential new restriction sites and double-click it. The program will list the potential new sites of the selected restrictase as cyan-highlighted position ranges just below the main program panel. Click the position of interest to change the sequence so that the desired new site is introduced. If you click on another position, the previous change will be reverted and the other site will get introduced. To make the change permanent, click the Apply & Process button. If you don't want to introduce any new site, click the Undo Changes button to cancel all recent changes. To save the modified sequence in a text file, click the Save Fasta button.