14.1 Find Restriction Sites

Restriction Enzymes¹ cut a nucleotide sequence at specific positions relative to the occurrences of the enzyme’s recognition sequence in the sequence. For example, the enzyme EcoRI has the recognition sequence GAATTC and cuts both the strand and the antistrand sequence after the G inside the recognition sequence² , leaving a single-stranded overhang (sticky end (overhang)):

To find and annotate restriction sites on a nucleotide sequence, go to Find Restriction Sites... under the Tools → Cloning menu or open the Restriction Analysis tab to the right of the sequence viewer.

You can configure the following options:

• Candidate Enzymes lets you select a set of restriction enzymes from which you want to draw the ones to use in the analysis. This includes the options to use commonly used, or all known commercially available restriction enzymes. If you have created your own restriction enzyme set from your local database then this will also be listed (see section 14.3 for how to create such a document). To select specific enzymes from a particular enzyme set, click the Advanced button (see below).
• Enzymes must match X to Y times: only returns restriction enzymes which cut the sequence X to Y times. Results for enzymes that cut the sequence more or less than this will be discarded. If you set X to be 0, when this operation is complete, it will report which candidate enzymes do not cut the sequence.
• Specifying cut regions: To specify a region of sequence where you want the enzyme to cut or not cut, choose one of the options below, and use the base counters to specify a sequence range that the options apply to. If you have selected a region of sequence in the sequence viewer, clicking the refresh arrow next to the base number counters will copy the selected region to this setting. The following options are available:
  • Cut Anywhere: Returns enzymes which cut anywhere in the entire sequence. It is not possible to select a subregion with this setting.
  • Must only cut between: Returns enzymes which only cut between the specified bases.
  • Must cut between (may cut outside): Returns enzymes which cut between the specified bases, and may also cut outside that region.
  • Must not cut between: Returns enzymes which only cut outside the specified bases.
• Advanced: This displays a table of all enzymes in your candidate set, including their recognition site, overhang, effective length and methylation information (Figure 14.1 ). Only the enzymes selected in this table will be considered in the analysis; initially, all rows are selected. You can click on the column headers to sort the table ascending or descending by that column, and you can Shift+click and Ctrl+click to select a range of rows and to toggle the selection of a row, respectively.

After configuring your options, click Apply to record the restriction enzyme site annotations on the sequence. The annotation shows the enzyme’s recognition site, cut site and methylation sensitivity. If Highlight Methylation Sites is selected, restriction sites which would not be cleaved due to the presence of methylation by Dam, Dcm or EcoKI are greyed out and marked with an asterisk (for further information on Highlight Methylation Sites see below).

Once the document is saved, two new tabs will appear above the sequence view: Enzymes displays the list of enzymes and their cut positions; Fragments displays a list of fragments that would be produced from the restriction digests. These tables can be exported as .csv files for subsequent processing with other software such as e.g. Microsoft Excel^®.

To select the region between two cut sites on a sequence, Shift+click on the two restriction site annotations in the sequence view.

To find enzymes that do not cut a particular sequence, use Find non-cutting enzymes under the Cloning menu. See section 14.3 for further details.

Highlight Methylation Sites

In Geneious Prime 2021 onwards, Geneious will flag restriction sites where restriction enzyme cleavage ability may be affected due to the presence of methyl groups added by methyltransferases Dam, Dcm, and EcoKI.

This is determined by experimental validation according to rebase.neb.com, which uses the following terms to describe the effect of methylation:

Restriction sites are greyed out and marked with an asterisk if the ability of the restriction enzyme to cleave at the restriction site is variable, impaired or blocked (see Figure 14.1 ). Methylated restriction sites where cleavage has not been experimentally validated, or might be methylated but have ambiguities in the target sequence, are highlighted with an asterisk but not greyed out.

The highlighted restriction sites and indicated methylation effect takes into consideration the neighboring sequence. For example, cleavage by XbaI is only blocked by dcm methylation if the recognition site (TCTAGA) is preceded by GA or followed by TC. If these flanking bases are not present, the site will not be marked as affected by methylation, but a note on the restriction site annotation will say what the effect of methylation would be if the correct bases were present.

Restriction Enzyme effective length

Effective length for restriction enzymes is displayed in both the Advanced table of enzymes, and in the Enzymes tab on the sequence viewer.

Effective length is a measure of how frequently an enzyme will cut, taking into account both sequence length and ambiguities. In other words, lower effective length means an enzyme is expected to cut more frequently. Because ambiguous bases are more likely to match a sequence by chance, they contribute less than 1 to the effective length.

Effective length is calculated as the sum of the following formula across all symbols in the recognition sequence, where n is the number of nucleotides each symbol represents.

1 - log(n) / log(4)

I.e. 1 for a ACGT, 0.5 for 2-ambiguity (MRWSYK),   .208s for 3-ambiguity (VHDB) and 0 for N.

Note: The sum displayed in Geneious is rounded down to nearest .0 or .5

See http://search.cpan.org/dist/BioPerl/Bio/Restriction/Enzyme.pm#cutter for a little explanation of why you would use this.