Ronna Hertzano, Aug 18th, 2003
Construction of fusion proteins with PCR (Overlap Extension PCR)
General considerations
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

All regular general considerations apply – staying in frame etc.
Eliminate the 'middle' stop codon if such a codon exists.
Depending on where your protein is targeted to, specifically if the fusion consists of 'parts of
proteins', it's important not to loose targeting signals (e.g. in the construction of transcription
factors dominant negative fusions – keep the nuclear localization signal (NLS)).
The basic premise
This is a simple two-step cloning technique. It is based on doing two independent PCR reactions with
partially matching overhangs followed by a third PCR reaction that will fuse the products of the first
two PCRs to one fusion DNA that would ultimately code for the desired fusion protein.
Assuming that you would like to fuse 'Protein A' amino terminal to 'Protein B'.
First two
PCR reactions
(use a PFU
polymerase
with blunt
ends)
Gene A
F
1
Gene B
R
2
F
3
R
4
Products of first PCR reactions
Third PCR
reaction
(use a PFU
polymerase
with Atailing or
tail on your
own at the
end, for TA
cloning for
sequencing)
Use 1µl of a gel extracted product from each of the first two PCR reactions
as a template for the third PCR reaction
During the denaturation and annealing, some of the template will anneal in a way that
will let the overhangs serve as 'primers' for DNA synthesis
After this happens, primers 1 and 4 serve as regular PCR primers and amplify the full
length fused product
F
1
R
4
The product of the third PCR reaction should be the fusion DNA
Ronna Hertzano, Aug 18th, 2003
Step 1:
Perform two independent PCR reactions:
For Gene A –
Forward primer:
regular. (primer 1 in the figure)
Reverse primer:
5'-reverse of the beginning of Gene B + regular reverse for Gene A-3'. (primer 2)
For gene B –
Forward primer:
5'- the end of Gene A + the beginning of Gene B -3'. (primer 3)
Reverse primer:
regular. (primer 4)
For Example:
if the end of gene A is 5'- AAA TCT GCT CTG GGA TCC – 3'
and the beginning of gene B is 5' – ATG CTG CTA GAA GCA – 3'
For the reverse primer for gene A, the fused primer will be:
5'- TGC TTC TAG CAG CAT GGA TCC CAG AGC AGA – 3'
For the forward primer for gene B, the fused primer will be:
5'- TCT GCT CTG GGA TCC ATG CTG CTA GAA GCA – 3'
The product of the two reactions should be:
1. Gene A + the first 15 nucleotides of Gene B.
2. The last 15 nucleotides of Gene A +Gene B.
It is best to use a PFU polymerase without A overhangs to generate these PCR products. It's easiest if
the template for these products is plasmid DNA (take ~50ng or plasmid as template). Gel purify the
PCR products, elute in 30-50µl. Run purified PCR product on a gel, to make sure that you have
excised the correct band. Use ~1µl of each product as the template for the next reaction.
Ronna Hertzano, Aug 18th, 2003
Step 2:
Perform a PCR reaction using ~1µl of each product as the template and primers 1 and 4. As you will
probably want to sequence the product of this reaction, it is easiest to subclone the product into a TA
vector and subclone it into the expression vector by restriction digestion after sequence verification. I
therefore recommend using a PFU enzyme that adds A overhangs (like the Easy-A enzyme) or to Atail the product after gel extraction and before the ligation.
note – elongation time of the third PCR should correspond to the size of the fusion product.
Gel purified
product of the
first PCR of
Gene A
Gel purified
product of the
first PCR of
Gene B
Take 1µl of each as a template for the third
PCR reaction;, run the PCR product, excise
the correct band and gel purify prior to
cloning into a TA vector for sequencing.
Good luck!!