Cloning in Silico¶

In this tutorial we are going to reproduce common steps required to perform cloning “in silico” experiments in Unipro UGENE: restriction analysis, digest into fragments, constructing new molecule.

Our task will be cloning a gene TC(R)4 from pBR322 molecule into antother vector pEGFP-N1.

Restriction Analysis¶

Let’s open the donor vector pBR322. Use file from $UGENE/data/samples/Genbank dir or download it manually using Access Remote Database (the Genbank Id is 208958)

At first we need to find suitable restriction sites to cut target gene.

Open the Restriction enzymes dialog and select restriction sites by the length of recognition equal 6.
Set Filter by number of results checked with min results = 1 and max results = 2.
Click OK.

As you can see the detected restriction sites are shown in the circular sequence view. You can choose manually the suitable sites.

We will use restriction sites EcoRI and AvaI that cut our target gene.

Digesting Into Fragments¶

Now let’s define fragment of new molecule.

Open Cloning ‣ Digest Into Fragments dialog. Here you need to select enzymes which will cut the active sequence into fragments.
Select AvaI and EcoRI sites by adding them to selected enzymes set.
Click OK to annotate the new fragments.

The newly created fragments are shown as annotations.

Each annotation describes fragment region and its terms.

Now we will digest vector pEGFP-N1 (CVU55762) into fragments.

Open the CVU55762 and activate Cloning ‣ Digest Into Fragments dialog.
We didn’t analyze any restrictions sites in this molecule, thus the option Use annotated regions for restrictions sites is disabled.
Instead we will use option Search for restriction sites to automatically search for required sites.
Click Search settings button and select single enzyme EcoRI. Then add it to selected enzymes and click OK

Single fragment of the CVU55762 will be created.

Constructing Molecule¶

The next step is the construction of new molecule.

Open Cloning ‣ Construct Molecule dialog. Here you will see available fragments from your current project.
Choose CVU55762 fragment 1 and click Add. The fragment will be added to new molecule contents.
Choose also pBR322 fragment 2. The new molecule will consist of these two fragments ligated.
As you can see the restriction sites on the right end of CVU55762 fragment and the left end of pBR322 are consistent (it is the same EcoRI site) and highlighted in green color.

Now let’s changes some settings of new molecule

Now set Make circular box checked. The other ends will become highlighted in red because of their inconsistency.
Click Force “blunt” overhangs to blunt the corresponding fragments end.
Click OK button to start ligation.

The newly created molecule will be opened. It will contain the gene TC(R)4.

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