DNAMAN provides many functions to help users performing restriction analysis. You may analyze restriction sites and draw a restriction map on a given sequence with a list of enzymes. You may also predict a restriction pattern on agarose gel when one or more DNA fragments are digested with restriction enzymes. You may use the electronic cloning function to generate new DNA constructs. The Silent Mutation and Directed Mismatch functions may help you to create or remove desired restriction sites on a target sequence.
The restriction site analysis function includes many features of enzyme cutting information on a DNA sequence. Choose the Restriction | Restriction Analysis command to perform restriction analysis on the current DNA sequence. A Restriction Analysis dialog box appears.
There are many options in the first page of Restriction Analysis:
1) Results: Show summary
Check this option to display the summary of restriction analysis in a text window. The summary includes the total number of enzyme cutting sites, a list of cutting enzymes with recognition sequences and their cutting positions, a list of cutting sites ordered by position and a list of non-cutting enzymes.
2) Results: Show sites on sequence
Check this option to display the sequence and cutting sites in a text window. The first letter of enzymes is located exactly on top of each cleavage base.
If this option is checked, you may check the With double-stranded sequence option to display double-stranded sequence with restriction sites.
3) Results: Draw restriction map
Check this option to display the restriction map in a Map window. You may modify the map with numerous tools as described in the Restriction Map section.
If this option is checked, you may check the With enzyme position to add enzyme positions with enzyme names on the restriction map. You may check also the With double-stranded sequence option to display double-stranded sequence in the map window. Check the Including annotations option to display annotations associated with the sequence in the map.
4) Results: Draw restriction pattern
Check this option to display the restriction pattern of the target sequence(s) digested with selected enzymes. The restriction pattern is shown in a graphic window. This option must be checked if you like to use the electronic cloning function.
5) Results: Ignore enzymes with more than
Check this option and enter a number in the behind box. If a restriction enzyme gives sites more than the number, DNAMAN will not show the enzyme.
6) Results: Ignore enzymes with less than
Check this option and enter a number in the behind box. If a restriction enzyme gives sites less than the number, DNAMAN will not show the enzyme.
7) Target DNA: Circular
Check this option if you want to correctly draw a restriction map or find restriction pattern of a plasmid sequence.
8) Target DNA: All sequences in channels
Check this option if you want to analyze and compare the restriction properties of all sequences in the channels. It is only activated when more than one channels contain DNA sequences.
9) Target DNA: dam methylation
Check this option if the DNA is dam methylated.
10) Target DNA: dcm methylation
Check this option if the DNA is dcm methylated.
For more information on the dam and dcm methylases and the affected restriction enzymes, choose the Info | Methylase command.
Press the Next button to go to the Enzyme Selection page. You may select:
-a restriction data file,
-a restriction enzyme list according to enzyme properties,
-restriction enzymes for analysis.
1) Selecting an enzyme data file
All available enzyme data files are listed in the Enzyme File box. Enzyme data files are stored in the DNAMAN system folder. DNAMAN provides two enzyme files, restrict.enz (180 enzymes) and dnamanre.enz (2524 enzymes). Select one enzyme data file in the Enzyme File box. You may save your own enzyme file from this dialog box. After selecting an enzyme list for restriction analysis, save the list by pressing the Save List button.
2) Selecting an enzyme list
You may shorten the selection list from a enzyme data file by restricting the cutting properties of enzymes.
DNAMAN provides two options Cutter and End that allow you to quickly select desired enzymes:
Cutter End
- All- All
- ≥ 5- Blunt
- ≥ 6- 5’ Overhang
- 3’ Overhang
Note that only A, C, G and T are considered for cutting length. For example, AccI “GT(A/C)(T/G) AC” is not in the list of “>=6 cutter”.
DNAMAN lists available enzymes according to these two options from the enzyme data file.
3) Selecting enzymes for restriction analysis
In the dialog box, the available enzymes in the enzyme data file are listed in left box. Select enzymes from the left to right enzyme box by double-clicking the enzyme names. You may select all the enzymes listed in the left enzyme box by clicking the Select All>> button.
To remove an enzyme from the selected enzyme box, double-click on the enzyme name. Clicking the Clear<< button removes all the enzymes from the right box.
You may select any number of enzymes for restriction analysis. However, if you have checked the Draw restriction pattern and All DNA in sequence channels options in the Restriction Analysis page, there is a limit of enzymes you can choose. If there are two DNA sequences in sequence channels, you cannot select more than two enzymes. If there are more than two sequences available in channels, you can choose only one enzyme for analysis.
DNAMAN provides two restriction enzyme data files: “restrict.enz” and “dnamanre.enz” which include 180 and 2524 restriction enzymes respectively. DNAMAN also allows users to create new enzyme files according to their needs.
Creating a custom restriction enzyme data file
1. Choose the Restriction | Restriction Analysis to display a dialog box and go to the Enzyme Selection page.
2. Select enzymes by double-clicking the enzyme names in the left box. For example, select the restriction enzymes listed in the puc18’s multiple cloning sites.
3. Click the Save list button to open the Open File dialog box.
4. Type a filename, e.g., puc18mcs.
5. Click OK. DNAMAN will add “enz” as the extension name. The new enzyme file name will be puc18mcs.enz.
Editing restriction data file
You can edit the default restriction enzyme file.
1. Choose the Restriction | Restriction Analysis to display a dialog box and go to the Enzyme Selection page.
2. Select the name of the restriction enzyme data file you want to edit from the Enzyme File box.
3. Click the Cancel button to exit. The selected data file will be the default data file.
4. Choose Info | Restriction Enzymes to open an Enzyme Information dialog box.
5. Click Edit button to open the enzyme data file. Follow the steps in page 13-2 to edit this file.