Restriction Comparator

Introduction

Restriction Comparator is a free software tool for comparative restriction analysis of two DNA sequences. It detects restriction sites that are either present in one sequence but not in the other or those with different frequencies. Furthermore, it provides a DNA digest electropherogram simulation displaying the restriction fragment patterns of the two sequences side by side. If you need to perform a more comprehensive restriction analysis of one DNA sequence, use the Restriction Analyzer app.

To perform the comparative restriction analysis, follow this workflow:

  • Provide two DNA sequences 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 Inputs

The DNA sequences to be analyzed can be provided by the user either directly - pasted into the sequence text areas, or in the form of text files. 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 text (not MS Word!), Fasta and GenBank format sequences are supported.

Settings

There are three parameters to be set before the analysis:

  • DNA form (linear or circular) - radio buttons located above the sequence text areas.
  • Restriction enzyme list - radio buttons in the "SETTINGS" panel located on the right side of the app window.
  • If the program should mask methylated sites - checkboxes at the bottom of the "SETTINGS" panel.

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 the recognition site and a presence of ambiguous positions in it. 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). In such cases you should choose the dam- and dcm- options.

The last section of Settings, "Discerning cleavage suggestions", allows you to modify the parameters of the search for restriction cleavage patterns suitable for distinguishing the input sequences from each other. "Target fragment range" is the range of fragment sizes where the discerning fragments will be searched for, i.e. for a cleavage pattern to be suggested as discerning, at least one fragment within the target range must be produced by such cleavage in at least one sequence. "Minimum fragment size difference" is a minimum required size percent difference between fragments, i.e. for a cleavage pattern to be suggested as discerning, at least one fragment within the target range must differ from each fragment from the other sequence by at least the entered number of percents of the size of that fragment. Restriction Comparator will try to find suitable discerning cleavage patterns using one restriction enzyme, two restriction enzymes, etc. up to the "Maximum number of enzymes to combine" set in the related input field. Numbers larger than 4 will be ignored and the value 4 will be used instead (analyzing all combinations of 5 or more enzymes is impracticable due to the required amount of time). The last option - "Prioritize these enzymes" - allows you to enter a specific set of enzymes the patterns of which should be displayed preferentially (sorted top) in the results list.

Understanding the Results

Results of comparative restriction analysis populate the three fields in the section "RESTRICTION SITES". Listings of sites corresponding with the names of the fields are arranged alphabetically row-wise except the last field where the different counts of each restriction enzyme cutting site detected in both sequences are listed in a table ordered alphabetically by restriction enzyme names.

Restriction digest fragments size calculation with electropherogram simulation is triggered by selecting restriction enzymes in the section labeled "RESTRICTION FRAGMENTS". There are three selection lists available, but more restriction enzymes can be selected when holding 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. The electropherogram image can be saved as a png file by clicking the "Save the image" pseudo-link below the image.

As the last type of output, Restriction Comparator screens the two input sequences for all possible restriction patterns and identifies those which could be used for unequivocal discrimination of one sequence from the other. The suggestions will be displayed after clicking on the items in the results summary for 1 enzyme, 2 enzyme-combination etc. in the "DISCERNING CLEAVAGE SUGGESTIONS" section at the bottom of the page. The virtual electropherograms can be clicked to display a detailed list of fragments produced by the particular restrictase combination.


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