Overlap Extension PCR (SOE) Tool
Fuse two or more amplicons by overlap extension — check the shared overlap and get the spliced product.
Paste at least two fragments, in the order you want them joined.
Learn more
What it does
Overlap extension PCR, also called splicing by overlap extension (SOE), fuses two amplicons without restriction sites or ligase. The inner primers add a shared block to the end of one fragment and the start of the next; after a few cycles the overlapping strands anneal and prime on each other, and the outer primers then amplify the full fusion.
How it works
The tool measures the longest exact match between the 3′ end of one fragment and the 5′ end of the next — the overlap that the two strands will anneal through. A junction passes when that block reaches your minimum; below roughly 15 bp the annealed duplex is too unstable to prime extension reliably.
Worked example
A 260 bp promoter fragment and a 230 bp coding fragment sharing 20 bp produce a 470 bp fusion. Reduce the shared block to 8 bp and the junction fails — in the tube that shows up as two separate bands and no fusion.
When to use it
Use it to fuse a tag to a gene, to join two exons, to create chimeric constructs, and to delete an internal segment by fusing the two flanking amplicons. If you are joining more than three fragments, a commercial assembly method is usually easier.
FAQ
- How much overlap does SOE PCR need?
- About 18–25 bp is typical. Below 15 bp the overlap melts at the annealing temperature and extension fails; beyond 30 bp you gain little.
- Do I need a special polymerase?
- A high-fidelity polymerase is strongly recommended, because the whole fusion product is amplified and any error introduced early is copied into the final product.
- Can I delete a segment with SOE PCR?
- Yes. Amplify the region upstream and the region downstream of the segment separately, giving them an overlap, then fuse the two — the segment between them drops out.
- Why do I get the two input bands but no fusion?
- Usually the overlap is too short or the two fragments were not gel-purified, so the outer primers have nothing to amplify. Check the reported overlap length first.