Overlap Extension PCR¶

What is Overlap Extension PCR?¶

Overlap Extension PCR fuses two or more DNA fragments that share short homologous overlaps at their ends:

Internal primers carry 5' tails that encode the desired overlap, which can be a spacer like in the picture below, or simply match the sequences to be joined.
After initial amplification, the overlapping fragments anneal and are extended to create the full-length product, which can then be amplified by outer primers.

Diagram showing overlap extension PCR process with internal primers carrying 5' tails

How to plan Overlap Extension PCR using OpenCloning¶

Like any other cloning method, click on the plus icon below a sequence in the Cloning tab and select Join Overlap Extension PCR fragments. Then, select the sequences to combine in the Assembly inputs field. OpenCloning checks adjacent fragments for sufficient terminal homology and proposes valid fused products.

Animation demonstrating Overlap Extension PCR workflow in OpenCloning

If you want to design primers for Overlap Extension PCR, see Primer design.