Virtual Subcloning

DNADynamo will place an 'Insert' sequence into a 'Vector' sequence and create a 'Construct' sequence. The 'virtual subcloning' tool is accessed by selecting 'Subclone into Vector' from the 'Vector' pull down menu. Note that you don't have to use this tool, you may find it easier to just copy and paste your insert sequence into your vector sequence manually.

How To Use This Tool

1) Displaying graphic maps containing the required restriction sites for the insert and vector

From the main window that contains the 'Insert' DNA sequence, select 'Subclone into Vector' from the 'Vector' pull down menu. Select the vector you wish to subclone into from the list on the left hand side of the display.

In the right hand side display, two graphic restriction enzyme maps are displayed. The insert is on top, the vector on the bottom. By default only the Multiple Cloning Site of the selected vector (as defined by its MCS Annotation/Vector Element) is shown, unless you uncheck the 'MCS Only' control.

Enzymes from the 'Dynamo' box are used to generate the restriction maps. In the current version of DNADynamo, the 'Dynamo' box is a subset of the 'uniqueSites' box that produce blunt or non-ambigious sticky ends. If you've updated from a previous version of DNADynamo that doesn't contain a 'uniqueSites' box (where the 'Dynamo' box is a subset of prototypes) you can press the 'RESET' button in the 'Enzyme Box' editor to update the predefined boxes. You can select additional appropriate enzymes manually in the Dynamo Box by opening the Restriction Enzyme box editor (Menu option Boxes->Enzyme Boxes). For example, the site/cut TT^CGAA is achieved by a group of 8 isoschizomers, with AsuII being the prototype. If you would prefer BstBI to be listed on the graphic maps, select BstBI in the Dynamo Enzyme box (Menu option Boxes->Enzyme Boxes - selected enzyme box -> 'Dynamo' --> select BstBI).

By default, only unique restriction enzyme sites are displayed. To add sites that appear twice in the sequence, select the 'Double Cutter' checkbox.

You can also opt to only display sites in the insert sequence outside the currently set ORF, using the 'outside orf' control.

2) Selecting the Restriction Sites to Join

First mouse over an enzyme name, and then click and hold the mouse button. Note that the enzyme is underlined as the mouse button is held and that any enzyme with a compatible cohesive end in the vector is also underlined.

Now drag the mouse, with the button still held down towards the vector. Note that a line appears between the selected enzyme and your mouse position. Move the mouse to the enzyme in the vector that you wish to ligate to. Note that this does not need to be a compatible cohesive end. When the vector site is selected, release the mouse leaving a line joining the two enzyme sites.

Repeat the procedure for the second pair of enzymes

. Finally, press the create button. A new window sequence window is opened containing the insert sequence placed into the vector sequence.

Note that if the ends to be joined do not represent two compatible cohesive ends or two blunt ends, DNADynamo fills in 5' overhangs to create a blunt end or chews back 3' overhangs to the blunt end and joins the two blunt ends together. If other behaviour is required you must copy and paste the DNA sequences yourself as required.

If the same site is used twice in the vector sequence (eg an insert flanked by two BamH1 sites placed into a single BamH1 site in a vector), the orientation of the insert is determined by the order in which you join the inserts sites. Connecting the 5' insert site first places in insert in the forward direction. Connecting the 3' insert site first places the insert sequence in the reverse orientation. In the tube of course you would have a mixture of the two orientations

The 'Vector Map' of the DNA Sequence will show the insert subcloned into the Vector. Selecting the 'Diagnostic' checkbox in the restriction enzyme site display table will show enzymes that cut at least once within the insert and once within the vector sequence. Pressing digest will give a comparison of fragment sizes that would result from digestion with the selected enzymes of the vector sequence alone or the vector and insert sequence.