Overlap Extension PCR
How do I simulate Overlap Extension PCR?
In this example we will use Overlap extension PCR to create an in-frame fusion between the SEC7 CDS and eGFP CDS
Open the Overlap Extension PCR Dialog
To open the Overlap Extension dialog, click menu Actions → Overlap Extension PCR....
Specify the Number of Fragments for Overlap Extension
By default the Overlap Extension PCR tool starts with two fragments
To set the number of fragments for Overlap Extension PCR, use the "+" or "–" buttons. The number of expected fragments are displayed in the Fragments tab, or listed in the "Fragments" dropdown menu.
For larger numbers of fragments, click the drop down and choose "Number of Fragments", in the dialog, enter the number of fragments then click OK.
Specify the Fragment Source Sequences
There are a number of ways to specify the fragments, you can use any combination of the following methods:
- First, set the Fragment via the "Fragments" dropdown menu.
- Click the dropdown to choose the "Source" of the fragment, or
- Click the "Click here" link to browse and set the "Source" of the fragment/s.
Drag and Drop
Drag and drop one or more source files into the Fragments panel.
If more than one sequence is added simultaneously then the "Order of Fragment" Dialog will automatically open. If required, use the "Reverse Fragment Order" button (with all or no sequences selected), or select sequences in the list and use the "Arrow" buttons to change the insert order.
Once the sequence order is set, click OK to continue.
Cut and Paste
First, set the Fragment via the "Fragments" dropdown menu, then copy a sequence from any compatible source file and paste to import it as the fragment source.
Set the Source for Fragment 1
In the "Fragments" tab, click the dropdown to show the Fragment 1 source sequence.
In Map view, click on a feature (if defined) to select the Fragment 1 region for the fusion. Alternatively, switch to Sequence view to manually select the fragment region.
Set the Source for Fragment 2
In the "Fragments" tab, click the dropdown to show Fragment 2 source sequence.
In Map view, click on a feature (if defined) to select the Fragment 2 region for the fusion. Alternatively, switch to Sequence view to manually select the fragment region.
Design Primers to Generate the Fusion
Switch to the "Fused Template" tab and click Choose Overlapping Primers....
Set the desired "Target Tm" for the "PCR primers" for each fragment, and for the "overlapping ends".
Click Choose Primers. Primers will be designed to amplify each fragment.
Name the Primers
Switch to Fragment View, use the "Fragment" dropdown to select each fragment in turn, and enter appropriate names for the newly designed primers.
Create the Overlap Extension PCR Product
Confirm the fragment arrangement and fusion are correct, switch to Sequence view if required to check the fusion at the nucleotide level.
Enter an appropriate name for the overlap extension PCR product.
Click PCR to create the fusion product.
Order Your Primers
In Primers view, select the new primers in the table, then click menu Primers → Export Primer Data.
At a minimum Primer names and nucleotide Sequence will be exported to a delimited text file.
Use the delimited text file to order the primers from an oligonucleotide synthesis service provider.