Fotia Microbiol. 44 (1), 11-14 (1999) www. b i o m e d , c a s .

c z / t a b u / f o l i a / i n d e x , ht,-

PCR Method for Generating Multiple Mutations

alnstitute of Microbiology, Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czech Republic
Fax +420 2 4447 1286
E-mail [email protected]
bDepartment of Genetics, Yale School of Medicine, New Haven, CT 06520, USA
Received June 9, 1998
Revised version September 14, 1998

AB.grP,ACT. Procedures to introduce point mutations, restriction sites and insert or delete DNA fragments are very important
tools to study protein function. We describe here two-step PCR-based method for generating single or multiple mutations,
insertions and deletions in a small region of the sequence. In the first step, a unique restriction site is introduced near the part
of DNA sequence to be changed, without changing the amino acid sequence. For this step, one of the methods already
described can be used. In the second step, mutations are introduced using mutagenic primers containing the unique restriction
site from the first step at the 5' end, paired with a universal primer crossing another unique restriction site present originaUy in
the sequence. The method is very simple, economic and rapid. In comparison with the traditional in vitro mutagenesis methods,
one can generate large numbers of mutated plasmids in hours.

The alteration of genes and/or proteins is particularly useful for studying the relations
between their structure and function. Site-directed mutagenesis is a fundamental tool of molecular bio-
logy and genetics. Various methods of in vitro mutagenesis have been described (Chert and Przybyla
1994; Ho et al. 1989; Kunkel 1985; Sang et al. 1996; Tomic et al. 1990; Zhong and Bajaj 1993) and are
widely used for introducing restriction sites, specifically modify coding sequences. PCR-based methods
are the most powerful tools, as they are generally quicker and more efficient than non-PCR-based
ones. On the other hand, some of the methods require two or more primers for each round of muta-
genesis, while others need a single very long oligonucleotide or two rounds of PCR for introducing one
mutation. Each of these increases the cost of mutagenesis.
Here we report a two-step PCR-based method which can be very efficient and economical in
those cases where a large number of nucleotide (amino acid) changes, deletions or insertions are to be
programmed in a small region of the sequence.
In our experiments, we changed 14 amino acids in a region of the 0t-subunit of the mitochon-
drial processing peptidase (MPP) from rat. The gene coding rat 0c-MPP was contained in the expres-
sion plasmid pGEMabinv (Striebel et al. 1996) which we routinely use for characterization of rat MPP.
The mutated region is part of a putative substrate binding site which is predicted to be a negatively
charged amphiphilic et-helix.

MATERIALS AND METHODS

All restriction enzymes used in this work and T4 DNA ligase were purchased from Boehringer
(Boehringer Mannheim, Indianapolis, IN, USA); they were used as recommended by the suppliers. For
sequencing, plasmid DNA was prepared using Qiagen Plasmid Purification Kit (QIAGEN Inc., Chats-
worth, CA, USA), and fragments were sequenced by the dideoxynucleotide chain-termination method
with a Sequenase TM DNA sequencing kit (United States Biochemical, Cleveland, OH, USA), both pro-
cedures were done according to manufacturer's protocol.
In our case, we were able to place a unique MluI restriction site in the middle of the negatively
charged amphiphilic 0c-helix region without changing the amino acid sequence. The new unique MluI
restriction site was introduced into plasmid pGEMabinv by using overlapping oligonucleotide primers
(P3 and P4) (Table I) which contain sequences that complement the template DNA sequence at the
3' end and complement each other at the 5' end. The sequences at the 5' end of the primers contain
a MluI restriction site which maintains the amino acid sequence of the expressed protein. Two PCR

*Correspondence author.
12 J. ADAMEC and F. KALOUSEK Vol. 44

products were made by using each of the internal overlapping primers (P3 and P4) paired with the cor-
responding external primers (P1 and P2) (Table I). The external primers P1 and P2 cross the natural

Table I. Sequences of oligonucleotides used

Primer Sequencea

P1 5'-CCAGACCTCGAGAGACACCAC
P2 5'-ACAGGACAAAACCGGTGCAGG
P3 5'-AGCCATACGCGTCATCTCAAT
P4 5'-GAGATGACGCGTATGGCTGTT

M1 5'-CATACGCGTCATCTCAATTTCCTCTTCTGTCAGGCGGGG
M2 5'-CATACGCGTCATCTCAATTTCATCATCTGTCAG
M3 5'-CATACGCGTCATCTCAATATCCTCATCTGT
M4 5'-CATACGCGTCATATCAATTTCCTC
M5 5'-CATACGCGTCATCTCAATTTCCTCATTTGTCAGGCGGGG
M6 5'-CATACGCGTCATCTCAATTTCCTGATCTGTCAG
M7 5'-CATACGCGTCATCTCAATTTGCTCATCTGT
M8 5'-CATACGCGTCATCTGAATTTCCTC
M9 5'-ATGACGCGTATGGC~GTTCAGTTTGA~CTTGAGGAC
M10 5'-ATGACGCGTATGGCTGTTCAGTTTGAACTTGATGACCTGAAC
Mll 5'-ATGACGCGTATGGCTGTTCAGTTTGAACTTGAGGAACTGAACATG
M12 5'-ATGACGCGTATGGCTGTTCAGTTTCAGCTTGAGGAC
M13 5'-ATGACGCGTATGGCTGTTCAGTTTGAACTTCAGGACCTGAAC
M14 5'-ATGACGCGTATGGCTGTTCAGTTTGAACTTGAGAACCTGAACATG

aUnderlined sequences indicate restriction enzyme sites. Double underlined letters indicate point muta-
tions that generate amino acid changes.

restriction sites XhoI and AgeI, respectively (Fig. 1). For both PCRs, 20 ng of plasmid pGEMabinv as
template and 20 pmol of each primer were mixed with 2.5 U Pfu polymerase (Promega, Madison, WI,
USA) in a 100 ~tL reaction containing 0.1 mmol/L each of the four deoxyribonucleoside triphosphates
(dNTP) and 1• Pfu Buffer (Promega). Thirty cycles of amplification were carried out with the follow-
ing conditions: I min at 94 ~ 1 min at 52 ~ 1 min at 75 *C. This was followed by final elongation step
at 75 ~ for 5 min. The product of PCR I or PCR II was then cleaved with MluI and XhoI or MluI and
AgeI restriction enzymes, respectively (Fig. 1). Digested fragments were separated on agarose gel elec-
trophoresis, extracted from gel slices using Qiagen Gel Extraction Kit (QIAGEN Inc., Chatsworth, CA,
USA), and ligated together with the pGEMabinv plasmid previously digested with XhoI and AgeI, to
create plasmid pGEMabMlu. After transformation of E. coli strain DH5cxF'TM (Life Technologies,
Grand Island, NY, USA) with ligated material (Nishimura et al. 1990), colonies were selected on LB
plates ( 1 % tryptone, 0.5 % yeast extract, 1.2 % bacto-agar, all Difco; 0.5 % NaC1) with ampicillin
(100 ~g/mL).
Mutagenic primers were synthesized to contain a sequence recognized by MluI at the 5' end
and a sequence introducing a mutation at the 3' end (Fig. 1). In PCR reactions we used mutagenic
primers M 1 - M 8 (Table I) paired with primer P1 or M 9 - M 1 4 with P2, respectively. Twenty ng of
plasmid pGEMabMlu was used as template for each PCR reaction in a total volume of 100/xL con-
taining 2.5 U Pfu polymerase (Promega), 1• Pfu Buffer (Promega), 0.1 mmol/L of the four dNTP's and
20 pmol of each primer. Thirty amplification cycles at 94 ~ for i min, 52 ~ for 1 rain and 75 *C for
1 min were carried out, followed by a further elongation step at 75 ~ for 5 min. The amplified frag-
ments designated F1-F14 were digested by enzymes MluI and XkoI (F1-F8) or MluI and AgeI
(Fg-F14), respectively, and separated on 1.2 % agarose gel. DNAs of the desired size were extracted
from gel slices by using Oiagen Gel Extraction Kit (QIAGEN) and ligated into plasmid pGEMabMlu
previously digested with enzymes MluI and XhoI for fragments F1-F8 or MluI andAgeI for F9-F14,
respectively, to reconstruct the mutant ~-MPP gene sequence in plasmid pGEMabMlu. After trans-
formation DH50cF''IM with the ligated material, colonies were selected on LB plates supplied with
ampicillin (100 ~g/mL).
1999 PCR METHOD FOR GENERATING MULTIPLE MUTATIONS A T ADJACENT SITES 13

r-~o ] Mlul
I r~ P1 ~ P4

P2* r
~PCR[ M/u! ~PCR[[ Agel
Xhol Mlu|
I I
I
Mlul AEel
t
I cleavedby ~ cleaved by
rXhol and Mlul Xhol and Mlul

[__
[

L J
~ ligated into
pGEMabinv/AEeI/Xhol

Mlul 7
r
Mlul

J I Z
Xho] ~ _ M1 Agel
M2
M3
P1 M4
M5

MIO
Mll . P2
M12~i~_ ~ 9
M13
M14 w -

Fig. 1. Principle of PCR method for generating multiple mutations at adjacent sites. Oligonucleotide primers are represented
by the arrows at their annealing sites in the target DNA sequences of plasmids pGEMabinv and pGEMabMlu. The two inter-
nal primers containing the unique restriction site MluI (P3, P4) anneal to opposite strands of the DNA template at the region
to be modified. The external primers (P1, P2) cross the natural restriction sites .XhoI and AgeI, respectively. PCR I (140 bp)
and PCR II (130 bp) were performed separately, their products were cleaved by.X7~oI and MluI orAgeI and M/ul, respectively,
and the fragments were ligated into plasmid pGEMabinv, previously digested by AgeI and XT~oI, to create plasmid
pGEMabMIu. To generate mutations, we used mutagenic primers containing the MluI restriction site paired with external
primers P1 (for M1-M8) and P2 (for M9-M14). The PCR fragments were then cleaved by appropriate restriction enzymes and
used to reconstruct mutant sequences. Changed amino acids are represented as: 9 = D ~ N, O = D ~ E, 9 = E ~ Q,
[] = E----~ D.
|4 J. ADAMEC and F. KALOUSEK Vol. 44

RESULTS A N D DISCUSSION

In the first step using the protocol described above we converted the plasmid pGEMabinv to
pGEMabMlu, which contained a unique MluI restriction site nearly in the middle of the segment
to be mutated. Four of five recovered plasmids were positive for the MluI site. Two plasmids,
pGEMabMlu-1 and pGEMabMlu-2, were then sequenced between the XhoI and AgeI sites to check
for mistakes from PCR amplification; none were found. In the second step, we generated the mutant
PCR fragments, F1-F14, as described, and ligated them into the cleaved plasmid pGEMabMlu-1 to
obtain the mutated plasmids pMutabl-pMutabl4, respectively. The cloned fragments were sequenced
to confirm the presence of the mutations and to verify that no undesired changes occurred in the
sequence during the PCR process. We did not detect PCR mistakes in any mutant plasmids and the
desired mutations were present in all but two clones, pMutab-7/2 and pMutab-10/1 (other pMutab-7
and pMutab-10 clones contained the mutations). We have also used this method successfully in other
similar mutagenesis projects (not shown).
Even though the cost of oligonucleotides depends on the distance between a unique restriction
site and the mutated sites, we found this method very rapid, simple, and efficient particularly for gener-
ating multiple mutations at the same or adjacent sites. Compared to the method using uracil incorpo-
ration in the template DNA (Kunkel 1985), we were able to generate all desired mutations in a much
shorter time. Generally, the time required for preparation of a large number of mutations was less than
3 d. Moreover, the very high efficiency (90-95 %) greatly reduces the number of colonies that need to
be screened (we test only 2 - 3 for each mutation), and the use of the high-fidelity Pfu polymerase
decreases the risk of unprogrammed mutations.

This work was supported by Fogarty International Research Collaboration Award TW0530 and Grant Agency of the
Czech Republic grant no. 204/98/0416.

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