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Restriction Mapping and Analysis

The powerful Digestor tool allows you to quickly and easily create restriction maps from your DNA sequences. In addition, the restriction analysis data can be organised so that you can instantly focus on the particular information you're seeking. The Digestor tool has such an incredibily simple and intuitive interface, that there is little to explain here that will not be immediately obvious.

Selecting Enzymes

On the current entry level PC (Pentium III 450), the restriction analysis algorithm is so fast that for sequences less than 50,000 bases, the digestion patterns of 70 of the most common restriction enzymes is automatically determined. For larger sequences (or slower computers), it is necessary to individually select the enzymes you wish to cut with, by ticking the checkbox in the left-most column, or just clicking the right mouse button and pushing Compute All Enzymes. The fragment pattern of the currently highlighted enzyme is automatically displayed on the Sequence Map, and the fragment sizes are displayed in the right-most column.

Multiple Enzyme Digests

To show the restriction pattern of multiple enzymes, simply hold down the CTRL key, and select the desired enzymes. The cutting positions will automatically appear on the Sequence Map and the calculated fragment sizes will be listed on the right-most column. The actual fragments can also be selected, and the corresponding location in the Sequence Editor will be highlighted, as will the region on the Sequence Map.

Sorting Restriction Analysis Data

By default, the restriction analysis data is sorted alphabetically by enzyme name. To sort the data by one of the other fields, simply click on the appropriate column heading. Clicking the same heading again will reverse the sort order. Sorting by number of matches, for instance, makes it very easy to identify an enzyme that cuts your sequence a particular number of times.

Digesting Sequences with Degenerate Characters

The Digestor tool can analyse DNA sequences that contain degenerate (ambiguous) characters. The algorithm recognises cuts 'pessimistically'. That is, it only records a cut when it is sure of the recognition site. For example, the recognition site, GAATTC will not match GAVTTC (where V = A or C or G) in the DNA sequence, however, the site AGVGA will match AGVGA, AGRGA, AGAGA, AGCGA and AGGGA. For more details about how Expression handles amiguity in sequences, see the Reverse Translation and Handling Ambiguous Characters tutorial.

Annotating Restriction Sites in Your Sequence

You can automatically annotate your sequence with the currently selected restriction sites, simply by right clicking and selecting Annotate. Once annotated you can move to other tools and still have the positions of the selected restriction sites conveniently marked.

Related Articles

Using the Sequence Map

Annotating Your Sequences

Reverse Translation and Handling Ambiguous Characters

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Restriction Mapping and Analysis

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