Overlap Extension PCR (a.k.a. Fusion PCR)
References:
Roche Expand Long Template Handout
Sambrook & Russel. “Site Specific Mutagenesis by Overlap Extension” Molecular
Cloning. CSH Protocols; 2006; doi:10.1101/pdb.prot3468
Purpose:
To amplify fused pieces of DNA from two separate sources. This protcol is particularly
handy for adding promoters or tags to a given gene sequence. The fused pieces can
easily be cloned into a vector for amplification and sequencing or transformed directly
into yeast. This protocol can also be modified to generate site specific mutations within a
given gene by engineering the desired mutation into both Primer2 and Primer3.
Primer Design:

Design Primer 1 and Primer 2 to amplify Gene1. Settings that I use for VectorNTI or
Primer3 are:
o 40 < Tm < 65
o 35 < %GC < 60
o 18 < Length < 25

Design Primer 3 and Primer 4 to amplify the tag. If Primer 3 will be the fusion
primer, design it to have 25 – 30 bp of homology to the sense strand of the 3’ end of
Gene1 + 20 – 25 bp of the sense strand of the 5’ end of the tag.

Check your primers well. Primer 3 and Primer 2 should have some reverse
complementation, but should not be identical.

If you are going to directly transform your fusion product into yeast, design Primer 5
& Primer 6 to have 30 bp of homology to the region of recombination.
Osborne, E.
06/2008
Protocol
Step 1: Amplify the two separate fragments

Set up two parallel PCR reactions to amplify Gene1 and the Tag. Use the Expand
Long Template PCR protocol as follows as an example of each reaction:
Reagent
1x
H2O
40.622 µl
10 x Expand Buffer + 17.5mM MgCl
5 µl
10 mM dNTP's
1 µl
100 μM Primer1 (or 3)
1 µl
100 μM Primer2 (or 4)
1 µl
(200 ng/µl) gDNA or
(50 ng/µl) Plasmid DNA
1 µl
5 U/μl Expand LT TAQ
0.378 µl
50

Run the PCR for 12 cycles on KO-TAG (below). This is typically not enough to see
a band on a gel. However, it is enough to start some products which will be the
templates in the Step 2 PCR reaction. Between the two PCR reactions, try to avoid
going over 30 cycles total or mutations will start to become a problem.
1.
2.
3.
4.
5.
6.
7.
8.
94C 2:00 min
94C 0:30 min
50C 0:30 min
68C 3:00 min***
Goto Step2 11x
68C 7:00 min
4C for ever
END
*** This time changes as your expected product length changes. Elongation time
= 1min/1kb fragment + 1 min

Clean up the fragments. Use a Qiagen PCR Purification Kit or a Qiagen Nucleotide
Removal Kit (if your fragments are <200 bp) to clean up the products. Elute in 50 µl
EB or H2O.
Step2: Fuse the two fragments

Using the two products from Step1 as the templates for a single PCR reaction, fuse
the two products into one. This reaction will either use Primer 1 and Primer 4 if the
products are to be cloned or Primer 5 and Primer6 for direct transformation.
Reagent
H2O
10 x Expand Buffer + 17.5mM MgCl
10 mM dNTP's
100 μM Primer1 (or 5)
100 μM Primer4 (or 6)
Gene1 fragment from Step1
1x
34.622 µl
5 µl
1 µl
1 µl
1 µl
4 µl
Osborne, E.
06/2008
TAG fragment from Step1
5 U/μl Expand LT TAQ
4 µl
0.378 µl
50

Run the PCR for 18 cycles on KO-TAG (see above).

If the final fragment is to be used in a TOPO TA cloning reaction, add 1µl of
AmpliTAQ GOLD TAQ Polymerase and allow the reaction to incubate at 72 C for
10 min.

Clean up the fused fragment. Run the entire 50 μl of the fusion reaction on a new
1 % agarose gel + EtBr (made with 1xTAE) in a gel box with fresh 1xTAE. Run the
gel to completion (100v, 1hr). Cut the bands and extract the DNA with a Qiagen Gel
Extraction Kit. Elute in 50 µl EB or H2O.
Future Directions

To clone fragments into a vector, use 2 µl of cleaned fusion product in an Invitrogen
TOPO TA Cloning Reaction. Tranform 2 µl of the cloning reaction into 50 µl
TOP10 e.coli cells.

To use fragments for direct transformation into yeast, use all 50 µl of cleaned fusion
product in the Manitoba Best Yeast Transformation Protocol.

Sequence your results to check for errors.
Troubleshooting
 No band?
o
o
o
The most common problem is that there is not adequate starting template in
Step1. Try to increase the amount of starting template.
If your fusion fragment is small, try to extend the number of cycles beyond
the 30cycles recommended, but be aware that mutations will increase as you
do so.
If your fragments are large and/or stubborn, run the PCR reactions in Step1
for 20 – 30 cycles and clone those products into a TOPO TA vector.
Sequence the vectors to find error-free constructs that can be used as starting
templates for Step1 amplification, this time with only 12 cycles. This works
well especially for amplifying large genes out of gDNA.

Too many bands?
o Try a higher annealing temp (higher than 50C) in both PCR reactions.

Lots of mutations?
o Minimize PCR cycles.

Only one of the fragments shows up at the end?
o This is usually an issue when Step1 amplifies a disproportionate number of
one of the fragments compared to the other. Try performing Step1 for 20 –
30 cycles and then running the products on a gel. Chances are, one product
is amplifying like crazy, while the other product is not well-represented.
Change the number of PCR cycles for each fragment in Step1 so that the two
fragments are about equal in the Fusion PCR reaction.
Osborne, E.
06/2008
Osborne, E.
06/2008