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SOE PCR (splicing by overlap extension PCR) joins two or more DNA fragments end to end by giving their ends a shared, complementary sequence. During PCR the overlapping ends anneal and prime each other, so the fragments fuse without restriction enzymes or ligase. It is also called overlap extension PCR, overlapping PCR or extension PCR.
Short answer
SOE PCR splices DNA by overlap. Each fragment is amplified with a primer whose 5′ tail matches the neighbouring fragment, so the 3′ end of one product can act as a primer on the other. A final amplification with the two outermost primers recovers the full-length joined product. Design choices that decide success are overlap length, overlap melting temperature, polymerase fidelity and the number of fragments.
Definition and scope
The method was described in US patent 5,023,171 (Ho and Horton, filed 10 August 1989, granted 11 June 1991, assigned to Mayo Foundation) as a way of splicing genes by overlap extension using the polymerase chain reaction. Google Patents lists its anticipated expiry as 10 August 2009. Later work describes the same principle as joining fragments through complementary overlapping ends, with no need for restriction sites or ligase (Zarghampoor et al., 2020).
Three names are used for essentially one technique:
SOE PCR and SOEing
SOE stands for splicing by overlap extension. The verb form, SOEing, describes joining fragments this way.
Overlap extension PCR and overlapping PCR
These are the names used when the emphasis is on the overlapping ends rather than on the splicing outcome. They describe the same reaction design.
Extension PCR
Extension PCR is another name for the same approach: fragments with shared ends anneal and extend each other, and outer primers then amplify the joined product.
How SOE PCR works in practice
The patent describes the mechanism in four moves. First, each starting molecule is amplified separately by PCR using junction primers that carry complementary 5′ tails. Second, the products are mixed, denatured and reannealed. Third, the matching 3′ ends act as primers for each other, and the polymerase extends them into one duplex. Fourth, the joined product is amplified with the outer primers. The worked example in the patent used fragments that share 15 homologous nucleotides.
A community protocol (OpenWetWare, checked 29 October 2026) describes the same logic with two complementary internal primers bridging the fragments, each with a melting temperature near 60 C against its own fragment and its partner, and outer primers that have no complementary region. It runs an overlap PCR without outer primers, then adds them for a second round, and recommends a proofreading polymerase for the extension step.
Conventional versus single-tube SOE PCR
Zarghampoor et al. (Avicenna Journal of Medical Biotechnology, 2020) compare the conventional two-step approach with a single-tube method for splicing several fragments. The table summarises the figures that paper reports.
| Feature | Conventional two-step SOE | Single-tube SOE |
|---|---|---|
| How fragments are joined | Pairwise joins, with gel purification between steps | All fragments assembled in one tube |
| Example cycling reported | 12 cycles with a 6 minute 50 C step, then 30 cycles (95 C 30 s, 55 C 45 s, 72 C 2 min) | 13 cycles with no primers, then external primers at 0.3 uM and 32 cycles |
| Fragments in the study | Hard beyond two fragments, especially with AT-rich overlaps | Three fragments; other groups reported up to four at once |
| Stated drawbacks | Smears, multiple bands or a weak main band; described as tedious, time-consuming and not cost-effective | Overlap melting temperatures should be the same or similar; mismatched overlaps led to a touch-up SOEing PCR |
Overlap design checklist
The patent’s example overlap is 15 nucleotides, but the 2020 study states that overlaps of 30 to 60 bp are typically added to the 5′ ends of primers, and its internal primers were 20 to 33 bp. The patent gives no general overlap length, so treat 15 nt as a historical example and not as a design rule. Points drawn from the 2020 paper:
- Add an overlap of roughly 30 to 60 bp to the 5′ end of the junction primers.
- Estimate the overlap melting temperature with the NEB Tm calculator or the formula Td = 4(C+G) + 2(A+T), and keep the overlaps for a single-tube assembly at the same or a similar value.
- Prefer a proofreading enzyme such as Pfu to Taq, because the authors note that Taq can introduce mutations and that repeated rounds add polymerase errors.
- Expect AT-rich overlaps and more than two fragments to be the hardest cases.
Where SOE PCR matters in lab work
Because the join is made by sequence overlap alone, no restriction site or ligase step is needed. A 1995 report in Molecular Biotechnology (Senanayake and Brian) applied the overlap extension approach to precise large deletions. For single-site changes made with primers alone, see the guide on site-directed mutagenesis. For the broader workflow of moving a fragment into a vector, see gene cloning.
Reaction setup, cycling basics and primer handling are covered in PCR protocol basics. Spliced products are normally checked by size, as described in agarose gel electrophoresis protocol basics, and primer specificity can be examined as covered in NCBI BLAST for primers.
Common misunderstandings
The overlap is the same as a primer-binding site
Not exactly. The overlap sits between two fragments, so that the 3′ end of each acts as a primer on the other. The outer primers are the ones that bind the ends of the final construct.
Any overlap length works
The 15-nt example in the original patent is not a general rule. The 2020 paper recommends 30 to 60 bp tails and matched overlap melting temperatures for single-tube work.
More fragments only means more cycles
The same paper reports that joining more than two fragments is difficult in the conventional approach, particularly with AT-rich overlaps, and that repeated rounds add polymerase errors.
The patent still restricts use
The foundational patent, US 5,023,171, has an anticipated expiry of 10 August 2009 according to Google Patents, so the original method is no longer under that patent.
Frequently asked questions
What does SOE PCR stand for?
Splicing by overlap extension PCR. It uses overlapping complementary ends to fuse DNA fragments.
Is overlapping PCR the same as SOE PCR?
Yes in practice. Overlapping PCR, overlap extension PCR and extension PCR are names for the same approach.
Does SOE PCR need restriction enzymes or ligase?
Not for the splice itself. The 2020 study states that fragments are joined through complementary overlapping ends without restriction sites or ligase.
How long should the overlap be?
The 2020 study states that overlaps are typically 30 to 60 bp, added to the 5′ ends of primers. The 15-nucleotide overlap in the original patent was a worked example, not a general rule.
Why use Pfu rather than Taq?
The authors suggest Pfu because Taq DNA polymerase may create mutations, which matters when several rounds of extension are used.
Sources
- Zarghampoor F et al. A Single Tube Overlap Extension PCR Method for Splicing of Multiple DNA Fragments. Avicenna J Med Biotechnol. 2020;12(1):37-43. https://pmc.ncbi.nlm.nih.gov/articles/PMC7035467 (checked 29 October 2026).
- US patent 5,023,171, Method for gene splicing by overlap extension using the polymerase chain reaction. https://patents.google.com/patent/US5023171 (checked 29 October 2026).
- OpenWetWare, PCR Overlap Extension (community protocol). https://openwetware.org/wiki/PCR_Overlap_Extension (checked 29 October 2026).
- Senanayake SD, Brian DA. Precise large deletions by the PCR-based overlap extension method. Mol Biotechnol. 1995;4(1):13.
