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Overlap Extension PCR Protocol

PCR Protocol for DNA assembly by PCR extension of overlapping DNA fragments.
PCR overlap extension is useful for DNA cloning and site-directed mutagenesis.

Here, you will find 2 different protocols

  • a standard protocol for performing overlap extension PCR
  • our Fast & Steep PCR protocol for overlapping DNA fragments
Jump to OE-PCR protocolsSDM Protocol Page

Overlap Extension Polymerase Chain Reaction

The overlap extension polymerase chain reaction (or OE-PCR) is a variant of PCR. It is also referred to as Splicing by overlap extension / Splicing by overhang extension (SOE) PCR. It is used to insert specific mutations at specific points in a sequence or to splice smaller DNA fragments into a larger polynucleotide.

Splicing of DNA Molecules

As in most PCR reactions, two primers are  per target DNA sequence are required. To splice two DNA molecules, custom primers are used to extend DNA ends that are to be joined. For each molecule, the primer at the end to be joined is constructed such that it has a 5′ overhang complementary to the end of the other DNA fragment. Following annealing when replication occurs, the DNA is extended by a new sequence that is complementary to the DNA molecule. Once both DNA molecules are extended in such a manner, they are mixed and a PCR is carried out with only the primers for the far ends. The overlapping complementary sequences introduced will serve as primers and the two sequences will be fused. This method has an advantage over other gene splicing techniques in not requiring restriction sites.

To get higher yields, some primers are used in excess as in asymmetric PCR.


OE-PCR utilized to splice two DNA sequences

Overview of Overlap Extension PCR

The use of PCR to splice two sequences together with primers that are complementary to both sequences. The dark blue & dark red lines represent the RNA Primers. The arrowheads represent the 3′ -OH ends of the chains. Credits

Why perform Overlap Extension PCR?

At Civic Bioscience, we often use OE-PCR to fuse multiple DNA sequences together for the purpose of:

  • Cloning :  a complete gene cannot be amplified from cDNA because specific sequence segments are too different in composition (i.e GC content) and are very difficult to amplify when the amount of target DNA is very low.
  • Site-Directed Mutagenesis : performing DNA mutagenesis using a 2-sided PCR overlap extension followed by ligation or seamless assembly into a linerized vector.
  • Multiple gene fusions, cassette or plasmid engineering : things can become quite complex when we engineer custom clones for our customers. Assembling 9 DNA fragments together by seamless assembly (i.e Gibson assembly) won’t work efficiently. But, if assembly by OE-PCR is used to put together fragments in groups of 3, then seamless DNA assembly using pEASY-Uni will become easy enough to get our clones rapidly. We use the Fast & Steep PCR protocol to accomplish this.
pEASY®-Uni Seamless Cloning and Assembly Kit
Easily assemble up to 5 DNA fragments with CU101 pEASY-Uni

Expected Results from Overlap Extension PCR used to join 2 DNA fragments of different sizes

Overlap Extension PCR for 3 different mutants

Standard Overlap Extension PCR Protocol

Standard PCR reaction setup

ddH2O : to 50 ul

5x buffer: 10 ul

dNTPs (2,5 mM) : 4 ul

F primer (10 uM) : 1 ul (0,2 uM) )(10 pmol)

R primer (10 uM) : 1 ul (0,2 uM) )(10 pmol)

Fragment 1 : 1 ul and equimolar to 2 and 3

Fragment 2 : 1 ul and equimolar to 1 and 3

Fragment 3 : 1 ul and equimolar to 1 and 2

FastPfu FLY : 1 ul (2.5 u)

Standard PCR Cycling

1-5 min denaturation at 95°C


15-30s denaturation at 95°C

10-30s annealing at optimal Ta

10-30s/kb (2-6 kb/min) at 72°C

2-10 min final extension at 72°C


Fast & Steep OE-PCR Protocol

Fast & Steep PCR reaction setup

ddH2O : to 50 ul

5x buffer: 10 ul

dNTPs (2,5 mM) : 4 ul

F primer (10 uM) : 2 ul (0,4 uM)(20 pmol***)

R primer (10 uM) : 2 ul (0,4 uM)(20 pmol***)

Fragment 1 : 20-30% of final volume and equimolar to 2 and 3

Fragment 2 : 20-30% of final volume and equimolar to 1 and 3

Fragment 3 : 20-30% of final volume and equimolar to 1 and 2

FastPfu FLY : 1 ul (2.5 u)

Fast & Steep PCR Cycling

2-5 min denaturation at 95°C

5-10x :

20s denaturation at 95°C

30s annealing at Ta of highest primer Tm

30s-60s/kb (2 kb/min) at 68°C

5 min final extension at 68°C

Why you should use the Fast & Steep PCR protocol for your next Overlap Extension PCR

Very Fast PCR Protocol

  • 15-45 min for 300 bp and 10 kb respectively.
  • PCR cycling time varies depending on template lenght and ramping rates.

Efficient DNA amplification

  • Incorporates up to 100% of primers in a very small amount of PCR cycles.
  • GC content and primer specificity may affect the efficiency.

Easy assembly of DNA fragments

  • Get your assembled DNA in 30 min, on average

Low error rate / High-Fidelity PCR

  • 5 to 10 PCR cycles is sufficient.
  • Limit the chance of introducing errors by limiting the amount of doublings and heating cycles, without compromising the DNA yield.

Using the fewest number of PCR cycles helps to avoid DNA depurination and deamination


⇒ Depurination involves the loss of purine bases forming abasic sites.

⇒ Depurination decreases at higher pH.

⇒ Depurination is independent of DNA sequence.

⇒ Heating DNA for 10 minutes at 100°C with pH 7.0 leads to about 1 apurinic site per 1000 base pairs.


⇒ Cytosine can be spontaneously deaminated to form uracil.

⇒ Cytosine in native DNA is estimated to deanimate with a rate constant of 10-10/sec at 70°C.

Examples of DNA assembly using PCR overlap extension

Results from PCR Success Story #22

Fast & Steep PCR to generate LEFT and RIGHT fragments

Left 1, 2 or 3 (1.3 kb) fragments, each amplified from their respective parent template containing only either one of the three mutations, and thee Right 123 (0.6 kb) DNA fragment, used to add a second and common mutation to any of the 1rst mutations, were amplified by Fast & Steep PCR.

PCR Setup :

ddH2O : to 35 ul
5x buffer: 7 ul
dNTPs (2,5 mM) : 2.8 ul
F primer (100 uM) : 0.14 ul (14 pmol)
R primer (100 uM) : 0.14 ul (14 pmol)
*Minicircle : 300 ng
FastPfu FLY : 0.7 ul (1.75 u)

PCR Cycling: 7 cycles (Ta = 62 °C)

Fast & Steep Overlap Extension PCR for 3 mutations

Fast & Steep OE-PCR 1.9 kb

Left 1, 2 or 3 (1.3 kb; 10 ul each) and Right 123 (0.6 kb; 10 ul) DNA fragments were fused into a 1.9kb DNA molecule under 30 min using Fast & Steep PCR.

PCR Setup :

ddH2O : to 40 ul

5x buffer: 8 ul

dNTPs (2,5 mM) : 3.2 ul

F primer (10 uM) : 1.6 ul (16 pmol)

R primer (10 uM) : 2 ul (16 pmol)

LEFT DNA : 10 ul

RIGHT DNA : 10 ul

FastPfu FLY : 0.8 ul (2 u)

PCR Cycling: 8 cycles (Ta = 57 °C)

Total run time: 26 min

Gel load : 2 ul / lane