Restriction Analyzer is a free software tool for comprehensive restriction analysis of a DNA sequence. It detects all present and absent restriction sites and presents the results both as tabular listings and graphical output (annotated sequence). Furthermore, it provides a DNA digest electropherogram simulation with unlimited number of restriction enzymes.
To perform the restriction analysis, follow this workflow:
- Provide the DNA sequence to be analyzed.
- Modify the program settings as required and click the Apply & Analyze button.
- See the results. If needed, perform an in silico restriction digest with electropherogram simulation.
Should the app settings be changed, click the Apply & Analyze button after that to update the results.
DNA Sequence Input
The DNA sequence to be analyzed can be provided by the user either directly - pasted into the sequence text area, or in the form of a text file. To import a DNA sequence from a file, choose the file with the Browse/Choose File button (the exact name of the button depends on particular browser and system local settings). Unformatted plain text (not MS Word!), Fasta and GenBank format sequences are supported. Alternatively, you can download a DNA sequence from the GenBank database. Just fill in the accession number (e.g. NC_012920) and click "Download". Expect that it takes some time, usually a few seconds, to send the download request and receive the sequence.
There are three parameters to be set before the analysis:
- DNA form (linear or circular) - radio buttons located underneath the sequence text area.
- Restriction enzyme list - radio buttons in the "Restriction sites selection" field.
- How the program should handle methylated sites - checkbox at the bottom of the "Restriction sites selection" field.
The DNA form choice will affect chiefly the calculations of restriction fragments, but wrong choice will also make a difference in the detection of restriction sites located across a plasmid formal start/end boundary.
Restriction enzyme list can be either pre-set by the app, selected with site length and degeneration criteria, or provided in full by the user. The first case is a manual selection of the most frequently used enzymes, typically those whose recognition sites are present in the multiple cloning sites region of commonly used plasmid vectors plus some other enzymes traditionally used for restriction analysis and molecular cloning. In the second case, you can select a subset of all commercially available enzymes based on the length of recognition sites and a presence of ambiguous positions in them. Lastly, if a specific set of restriction enzymes is to be used in the analyses, one can either enter the list (paste it in the text area in the "Enter the restriction enzyme list" dialog) or load a list of enzymes provided as a plain text file with one restriction enzyme name per line.
DNA methylation blocks cleavage by some restriction endonucleases. The methylation pattern can be predicted from sequence and methylation sensitivity of individual restrictases has been described. The app thus can mask sites that will not get cleaved unless the plasmid DNA has been prepared from methylase-deficient bacteria or comes from a different source (e.g. a PCR amplification product). Proper setting of the "ignore sites blocked by methylation" option is needed for correct restriction sites detection.
Understanding the Results
Results of a restriction analysis populate several different data fields. After a succesful analysis, the table located on the right side of the window will dislay frequencies and cutting positions of all sites included in the analysis. By default, the sites are ordered ascendingly by frequency, but the order can be switched to descending or to alphabetical order with the arrows located just above the column labels. Placing mouse cursor over a restriction enzyme name will display its cutting pattern, isoschizomers, neoschizomers and a list of all restrictases producing compatible cohesive ends (isocaudomers).
There are three fields in the section "RESTRICTION SITES OVERVIEW" - Present, Absent and Unique. Listings of sites corresponding with the names of the fields are arranged alphabetically row-wise.
The section "ANNOTATED SEQUENCE" shows all the present restriction sites marked along the sequence. Placing mouse cursor over a site label will highlight the cutting pattern of the enzyme and will list positions of all sites cut by the enzyme with the present position highlighted with yellow color.
Restriction digest fragments size calculation with electropherogram simulation is trigered by selecting restriction enzymes in the section labeled "RESTRICTION FRAGMENTS". There are three selection lists available, but more restriction enzymes can be selected when pressing Ctrl key on the keyboard. Restriction enzymes can be removed from the present selection either one by one with another click while holding the Ctrl key pressed or all at once by selecting "none" at the top of the list. Note that the "RESTRICTION FRAGMENTS" section has its own settings of DNA methylation status that needs to be properly set for correct calaculations. The electropherogram image can be saved as a png file by clicking the "Save the image" pseudo-link below the image.