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 Sequences Containing the Fragments to be Joined

Locate and open sequences that contain the regions to be joined by overlap extension PCR.

Ensure the foremost window contains the leftmost part for overlap. This file will then be automatically selected as source for input Fragment 1 when you run the Overlap Extension PCR tool.

Start the Overlap Extension PCR Tool

Click menu Actions → Overlap Extension PCR → Overlap N fragments, where N is the number of fragments to join. In this example we will overlap 2 fragments.

Set the Source for Fragment 1

In the "Fragment 1" tab, Set the "Source of Fragment 1" file.

In Map view, click on a feature (if defined) to select the Fragment 1 region for the fusion. Alternately, switch to Sequence view to manually select the appropriate region.

Set the Source for Fragment 2

Switch to the "Fragment 2" tab, then set the "Source of Fragment 2" file.

In Map view, click on a feature (if defined) to select the Fragment 2 region for the fusion. Alternately, switch to Sequence view to manually select the appropriate 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", then click Choose Primers. Primers will be designed to amplify each fragment.

Confirm the fragment arrangement and fusion are correct, switch to Sequence view if required to check the fusion at the nucleotide level.

Click PCR, to simulate fragment PCR and overlap extension PCR, to create the fusion product.

Check the Fusion Product

Confirm the product is as expected in Map view.

Switch to Sequence view and confirm expected fusion is correct and in-frame.

Name your primers. Switch to Primer view, double click on each primer in turn and edit the primer details.

Click Save to save the new Fusion product file.

Order Your Primers

In Primers view, select the new primers in the table, then click menu Primers → Export Selected 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.