Jan 28, 2025
Overlap extension PCR
Forked from Overlap extension PCR
This protocol is a draft, published without a DOI.
- 1Institute for Synthetic Microbiology
Protocol Citation: Anna Behle 2025. Overlap extension PCR. protocols.io https://protocols.io/view/overlap-extension-pcr-dykx7uxn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 27, 2025
Last Modified: January 28, 2025
Protocol Integer ID: 119159
Abstract
Linear assembly of PCR fragments.
Can be used to quickly and efficiently fuse promoters, terminators, fusion proteins etc. without time-consuming sub-cloning steps.
Materials
STEP MATERIALS
Q5 High-Fidelity DNA Polymerase - 100 unitsNew England BiolabsCatalog #M0491S
Gel and PCR Clean-up kit Macherey and NagelCatalog #740609.250
Protocol materials
Q5 High-Fidelity DNA Polymerase - 100 unitsNew England BiolabsCatalog #M0491S
Gel and PCR Clean-up kit Macherey-NagalCatalog #740609.250
Before start
Before starting, thoroughly design and plan your experiment in silico, ideally using cloning software such as SnapGene.
Make sure all primers anneal only once in the actual template (e.g. when using a whole genome) and separate parts are able to anneal to each other after PCR.
I recommend going through the whole cloning process in silico prior to ordering primers to avoid mistakes.
Primer design
Primer design
Before starting, thoroughly design and plan your experiment in silico, ideally using cloning software such as SnapGene.
Make sure all primers anneal only once in the actual template (e.g. when using a whole genome) and separate parts are able to anneal to each other after PCR.
I recommend going through the whole cloning process in silico prior to ordering primers to avoid mistakes.
Design primers for all fragments. Primers for overlap extension PCR are designed in a similar manner as for Gibson assembly, except that the outermost parts do not contain overhangs to each other.
- Annealing parts of primer: Solid arrow in scheme; Should bind to template only once with a Tm of 55-65 ˚C
- Overhang parts of primer: Dotted arrow in scheme; total overlap between two fragments should have a Tm of at least 55 ˚C to avoid unspecific binding
Note
It can be beneficial to already add overhangs to the desired vector backbone to the primers flanking the fused part (i.e., P1 and P6 in the scheme) to facilitate downstream cloning.
This method will yield a linear fragment.
PCR of parts
PCR of parts
Prepare separate PCR reactions of each part that needs to be assembled.
Note
Since the primers contain long overhangs, it might be necessary to try different annealing temperatures. I generally recommend starting with the annealing temperature corresponding only to the part of the primer which anneals to the template and disregarding the overhang for now.
Q5 High-Fidelity DNA Polymerase - 100 unitsNew England BiolabsCatalog #M0491S
| A | B | |
| Component | Amount [µL] | |
| 5x Q5 buffer | 5 | |
| 5x High GC buffer | 5 | |
| dNTP mix, 10 mM each | 0.5 | |
| Primer fwd, 10 µM | 0.5 | |
| Primer rev, 10 µM | 0.5 | |
| template DNA | 0.5 | |
| Q5 Polymerase | 0.25 |
Gel-purify fragment of correct size.
Gel and PCR Clean-up kit Macherey and NagelCatalog #740609.250
Overlap PCR
Overlap PCR
- Prepare PCR mixture, without primers. Instead of a template, add your PCR parts. Use a large volume, i.e. 1/2 to 3/4 of the total PCR reaction. Make sure to use a molar ratio of ~1:1.
Note
A large volume can be beneficial because often, the concentration of gel-exctracted PCR fragments is low. I recommend using 50 ng of the larger PCR fragment. This step can be varied, but overloading the template DNA can lead to more unspecific product.
Example using Q5-Polymerase:
| 5x buffer | 5 µL | |
| 5x high GC buffer | 5 µL | |
| dNTPs | 0.5 µL | |
| template | 4 µL fragment 1 + 5 µL fragment 2 + 5 µL fragment 3 | |
| Q5 Polymerase | 0.5 µL | |
| H2O | to 24 µL |
- Run your PCR at 15 cycles, using the annealing temperature of the homologous regions.
- Remove PCR from cycler. Immediately proceed with Step 4.
Extension PCR
Extension PCR
- Add the two primers flanking the outer parts as you would in a normal PCR.
- Rerun your PCR at 30 cycles, this time using an annealing temperature matching your flanking primers.
- Important: Gel-extract your overlap extension product, as this method can result in non-specific side products!
- PCR reaction will likely yield multiple bands (e.g. the fragments you started out with), as well as a smear around the desired band.
This method, when successful, yields a strong band of the correct size that can be used downstream for standard cloning methods such as Gibson Assembly.