Overlap extension polymerase chain reaction

After further PCR cycles, to amplify the DNA, the sample can be separated by agarose gel electrophoresis, followed by electroelution for collection.

Efficiently generating oligonucleotides beyond ~110 nucleotides in length is very difficult, so to insert a mutation further into a sequence than a 110 nt primer will allow, it is necessary to employ overlap extension PCR.

In addition, the combination of OE-PCR and asymmetric PCR could be used to improved the efficiency of site-directed mutagenesis.

This is possible since OE-PCR relies on the utilization of complementary overhangs to guide the scarless splicing of custom DNA fragments in a desired order.

First, individual DNA sequences are amplified by PCR from different templates and flanked with the required complementary overhangs.

Eventually, outer primers targeting the external overhangs are used and the desired DNA product is amplified in the final PCR reaction.

The overall success of OE-PCR based DNA assemblies relies on several factors, being the most relevant ones the instrinsic features of the DNA sequence to assemble, the sequence and length of the overlapping overhangs, the design of outer primers for the final amplification and the conditions of the PCR reaction.

This image shows how OE-PCR might be utilized to splice two DNA sequences (red and blue). The arrows represent the 3' ends
This image shows how OE-PCR might be utilized to delete a sequence from a DNA strand
The image depicts the 3 main steps of OE-PCR Reaction.