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. To find enzymes that do not cut a particular sequence, use Find non-cutting enzymes under the Cloning menu. To select the region between two cut sites on a sequence, Shift+click on the two restriction site annotations in the sequence view. csv files for subsequent processing with other software such as e.g. 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. 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). The annotation shows the enzyme's recognition site, cut site and methylation sensitivity. 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.Īfter configuring your options, click Apply to record the restriction enzyme site annotations on the sequence. Only the enzymes selected in this table will be considered in the analysis initially, all rows are selected. Must not cut between: Returns enzymes which only cut outside the specified bases.Īdvanced: This displays a table of all enzymes in your candidate set, including their recognition site, overhang, effective length and methylation information. Must cut between (may cut outside): Returns enzymes which cut between the specified bases, and may also cut outside that region. Must only cut between: Returns enzymes which only cut between the specified bases. It is not possible to select a subregion with this setting. The following options are available:Ĭut Anywhere: Returns enzymes which cut anywhere in the entire sequence. 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. 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 set X to be 0, when this operation is complete, it will report which candidate enzymes do not cut the sequence. Results for enzymes that cut the sequence more or less than this will be discarded. To select specific enzymes from a particular enzyme set, click the Advanced button (see below).Įnzymes must match X to Y times: only returns restriction enzymes which cut the sequence X to Y times. If you have created your own restriction enzyme set from your local database then this will also be listed. This includes the options to use commonly used, or all known commercially available restriction enzymes. under the Tools → Cloning menu or open the Restriction Analysis tab to the right of the sequence viewer.Ĭandidate Enzymes lets you select a set of restriction enzymes from which you want to draw the ones to use in the analysis. To find and annotate restriction sites on a nucleotide sequence, go to Find Restriction Sites. The restriction enzyme information included in Geneious Prime was obtained from Rebase, available for free at. 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 sequence, leaving a single-stranded overhang ( sticky end (overhang)): Restriction Enzymes cut a nucleotide sequence at specific positions relative to the occurrences of the enzyme's recognition sequence in the sequence. The following sections give more detail on each option. You can also create enzyme lists, find restriction sites on your sequence of interest, and simulate digestion and ligation reactions. The cloning features in Geneious Prime allow you to simulate several different types of cloning, including Restriction cloning, Golden Gate cloning, Gibson Assembly, Topo cloning and Gateway cloning.
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