Mutagenesis Series: Overlap PCR

By TT -

 Introduction

PCR is a tool for used to multiply specific fragments of DNA, as an alternative to gene cloning,  that can be used to create gene variations in the laboratory. Overlap extension PCR is a technique that is commonly used for cloning large complex fragments, and fusing two gene fragments together. 

In this series, Overlap PCR is used to fuse two gene fragments together.  As mentioned in previous postings.  The KanR gene from E. coli will be used to tag D. radiodurans and block the ZWF gene. The protocol used for this process uses touchdown PCR cycling and is completed in 2 steps. The first step was completed separately.  This posting will go over the 1 step of the Overlap procedure that was completed. 

Procedure

This protocol was optimized to eliminate the need for optimizing PCR cycling conditions.  There are no specific temperatures that must be used for the annealing cycle and the temperatures decrease on a gradient of 0.5 degrees Celsius per cycle.  

This step was completed in two reactions.  This step required 22.5 uL of Q5 master mix, 100ng/kb of DNA, and 21.5 uL of PCR water.  In tube 1, the left fragment which contains D. radiodurans with primers 1 and 2, and the middle fragment which contains the KanR plasmid with primers 3 and 4, were added together, as one reaction.  In tube 2, the right fragment which contains D. radiodurans which contains primers 5 and 6, and the middle fragment which contains the KanR plasmid with primers 3 and 4, were added together, as one reaction.  No additional primers were added in this step as they were not required beyond the original PCR application. 

These two reactions were put into a thermocycler using the touchdown method pictured below, for 15 cycles. 



Results

Gel of step 1 of Overlap PCR without primers


As there were no additional primers added to these reactions, there was nothing to show on a gel with a 1kb ladder because the fragments were not yet fused together.  They were merely amplified.  The next step will be the final fusion step of this protocol.  That step should result in a 2-way assembly by fusing together the overhang sections created by the primers that will be added. 



References

Hilgarth, R. S., & Lanigan, T. M. (2019). Optimization of overlap extension PCR for efficient transgene construction. Elsevier. https://doi.org/10.1016/j.mex.2019.12.001

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