Overlap extension polymerase chain reaction



The overlap extension polymerase chain reaction (OE-PCR) technique was first published in 1988 by Higuchi et al. and describes a process based on the polymerase chain reaction used to a) insert mutations at specific points in a sequence, further than ~55 nucleotides from either end and/or b) produce polynucleotides from smaller fragments.

To insert a mutation into a sequence, a primer is generated depending on requirements. A single substitution may be included or a totally new sequence added at the 5' end. If a deletion is required, then the continuation of the sequence 5' of the deletion effectively becomes an addition. The 3' end must be complementary to the template strand so that DNA polymerase can copy the template from the primer (DNA polymerase can only copy DNA in one direction; 5' to 3').

Once the primer has annealed to the template, the DNA polymerase proceeds to the end of the template. The duplex is denatured and the second primer anneals to the newly formed DNA strand, containing the first primer. Replication proceeds to produce a strand of the required sequence, containing the mutation.

The duplex is denatured again and the first primer can now bind to the latest DNA strand. The replication reaction continues to produce a fully dimerised DNA fragment. 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 OE-PCR the sequence being modified is used to make two modified strands with the mutation at opposite ends, using the technique described above. After mixing and denaturation, the strands are allowed to anneal to produce three different combinations as detailed in the diagram. Only the duplex without overlap at the 5' end will allow extension by DNA polymerase in 3' to 5' direction.

Following separation, the eluted fragments of appropriate size are subject to normal PCR, using the outermost primers used in the initial, mutagenic PCR reactions.