CHAPTER 14

PCR-Based Site-Directed Mutagenesis

Atsushi Shimada

1. Introduction
Protocols for site-directed mutagenesis are widely used in molecular
biology and include many polymerase chain reaction (PCR)-based meth-
ods that have been developed in order to achieve efficient mutagenesis
of a target DNA sequence (1-4). However, some of these methods
described require two or more specific-oligonucleotides for each round
of mutagenesis, making the cost of such procedures expensive. This
chapter describes an efficient and economic PCR-based site-directed
mutagenesis method, which is designed to introduce a series of muta-
tions into DNA cloned into pUC vectors (pUC 18, 19, 118, 119). The
protocol uses a combination of a primer designed for introducing a muta-
tion at the target sequence, with primers that may be reused for each
mutagenesis reaction (Fig. 1). By using this method, a series of site-
directed mutations may be undertaken that only require a single primer
for each desired change, and furthermore, no reiterative transformation
steps are necessary.
2. Materials
1. Taq DNA polymerase, 5 U/)aL (TaKaRa Shuzo Co. Ltd., Kyoto, Japan).
2. lOX PCR buffer: 100 mMTris-HCl, pH 8.3, 500 mMKCl, 15 mMMgClj,
0.01% (w/v) gelatin.
3. dNTP Stock: 2.5 xnM each of dATP, dCTP, dGTP, and dTTP (TaKaRa
Shuzo).
4. Oligonucleotides: MUT 1-6 (5 pmol/|aL), M13 M4 (5 and 20 pmol/fiL),
M13 RV (5 and 20 pmol/^L), and Rl (5 pmol/|aL) (see Fig. 2 on p. 160).

From Methods in Molecular Biology, Vol 57 In Vitro Mutagenesis Protocols

Edited by M K Trower Humana Press Inc , Totowa, NJ

157
158 Shimada

multi-cloniiig iiic

MBI>ninerM4

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m '-A.

I- M I 3 PrimerM4

[3]

MI3 Primer RV

[4]

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Fig. 1. Principle of PCR in vitro mutagenesis. [ 1 ] First round PCR of target

DNA following cloning of sequence into multicloning site of one of the pUC
series of vectors. One of the MUT primers is chosen to destroy a restriction site,
based on both the direction of Rl primer (primer for introducing a mutation)
and the restriction site used for cloning the target DNA sequence. The first
round PCR is carried out by the combination of R1 primer and M13 primer RV
(or M13 primer M4), MUT primer, and MI3 primer M4 {or Ml3 primer RV)
PCR-Based Site-Directed Mutagenesis 159

MUT 1 5' CATGATTACGAGTTCTAGCT 3'

MUT 2 5' GATCATCTATAGTGGACCTG 3'
MUT 3 5' TGATTACGCCTAGCTTACAT 3'
MUT 4 5' GGCCAGTGCCTAGCTTACAT 3'
MUT 5 5' CAGGTCCACTATAGATGATC 3'
MUT 6 5' ACGGCCAGTGAGTTCTAGCT 3'
M13 M4 5' GTTTTCCCAGTCACGAC 3'
M13 RV 5' CAGGAAACAGCTATGAC 3'
For the design of Primer Rl,see Note 1.
5. Plasmid DNA, into which the target sequence is cloned, prepared using
standard protocols (5).
6. Phenol saturated in TE buffer.
7. Chloroform.
8. 3M Sodium acetate, pH 4.8-5.2.
9. Appropriate restriction enzymes.
10. SUPREC-01. Filter cartridge for rapid recovery of DNA from agarose gels
(TaKaRa Shuzo).
11. SUPREC-02. Filter cartridge for rapid purification and/or concentration of
DNA samples (TaKaRa Shuzo).
12. T4 DNA ligase (350 U/|LIL) with lOX reaction buffer (660 mM Tris-
HCl, pH 7.6, 66 mMMgCl2, 100 mMDTT, and 1 mMATP) (TaKaRa
Shuzo).
13. Corcvpetent Escherichia coli cells.
14. TE Buffer: 10 mMTris-HCl, pH 8.0, 1 mM EDTA.
3. M e t h o d s
The procedure requires that the target DNA sequence is cloned into a
pUC vector (pUC 18, 19, 118, 119).

separately in two tubes. [2] After DNA purification to remove excess primers,
the amplified products are mixed, heat denatured, and annealed. [3] Taq DNA
polymerase is added to complete the DNA heteroduplex. [4] The second round
PCR is performed using M13 primer M4 and Ml3 primer RV flanking oligo-
nucleotides, which will result in two types of amplified products, (a) and (b).
[5] The amplified products are digested with two restriction enzymes, one of
which shall recognize the site (X) that had been destroyed by the MUT primer
and the other which recognizes the appropriate site (Y) within the multicloning
site. [6] Redone the digested fragment into the vector digested with the same
two restriction enzymes. Only the fragment (a) that contains the mutation intro-
duced by Rl primer sequence will be recloned.
160 Shimada
O pUC 18(+) strand
—CACGAAACAGCTATCACCATGATTACCATGATTAC|GAATTCJ GAGCTC| GGTA |CC| CGG |GJ GATCC| TCTAGA|
~ ~ ~ ~ ^ EciM I Sac I Kpn I Sma 1 BamH i Xba 1
MUPnmerRV ' „„„,
MUT 1 G T— ^ ^""''
MUT2 A T-

GTCGACl CTCCAOI GCATCCl AACCTT GGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAAC-

Son Pstl Sphl Wind 111 -^
Ace I M13PnmerM4
HincII •^—A T MUT 4
—G-
-C MUT 5

OpUC 19(+) Strand

5'"~CAGGAAACAGCTATGACCATGATrACGCC | A A 0 C T T | G C A T G C | C T G C A G | G T C G A C | T C T A G A | G G A T C |C| C C G | G G |

• HmdIII Sphl Psil Sail Xbal BamH I Sma I

MUT 3 T A-*. ^,^„
MUT 5 C A T—•
•^ G T A MUT 2

TACC| G A G C T C | GAATTCJ ACrGGCCGTCGTnTACAACGTCGTGACTGGGAAAAC——""3'

Kpnl Sad EcoRl -^
M13PnmerM4
-«—T G MUT6

Fig. 2. Position of MUT primers, Ml3 primer M4 and RV. The MUT
primers are designed to anneal within the multicloning site of pUC vectors;
each has a single base mismatch in the recognition sequence of the restric-
tion enzymes with which it is associated. M13-M4 and M13-RV primers
also bind to sequences within the multicloning site, but flank the MUT series
of oligonucleotides.

3.1. Preparation
of First Round PCR Products
Design and prepare the mutagenic primer (Rl) to introduce the desired
change into the target DNA sequence (see Note 1).
Prepare the first round of PCR reactions using the primer combinations
described in a and b (see Fig. 3 and Note 2):
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a. Combination of Rl primer and Ml3 primer M4* (or Ml3 primer RV):
10 pg—1 ng plasmid template DNA, 1 )iL Rl primer, 1 i^L M13 primer
(5 pmol/nL), 10 |xL lOX PCR buffer, 8 |iL dNTP stock, 0.5 |uL Taq
DNA polymerase, make up to 100 nL with distilled sterilized water.
b. Combination of a MUT primer (MUX 1-6) and M13 primer RV* (or
M13 primer M4). 10 pg-1 ng plasmid template DNA, 1 jxL MUT
primer, 1 (xL M13 primer (5 pmol/|j,L), 10 ^L lOX PCR buffer, 8 ^L
dNTP stock, 0.5 ^iL Taq DNA polymerase, make up to 100 |aL with
distilled sterilized water.
3. Overlay each tube with mineral oil (approx 100 |j,L) and perform first stage
PCR using the following program for 25 cycles: denaturation, 94°C for 30
s, primer annealing, 55°C for 2 mm; primer extension, 72°C for 1 mm (see
Note 3).
4 Remove a small aliquot (5 |j,L) from 2a and 2b for analysis by agarose gel
electrophoresis (1.5% gel) to determine if the PCR reactions were success-
ful (see Note 4).
5. Purify the PCR products using standard phenol-chloroform extraction/
ethanol precipitation. Alternatively, products available commercially such
as SUPREC-02 filter cartridges may be used. Each of the purified PCR
products should be resuspended in 100 |iL TE.
3.2. Heteroduplex Formation
and Second Round PCR
1. Prepare the following reaction mixture and then overlay with 100 ^iL of
mineral oil: First round PCR product a, 1 )iL; first round PCR product b,
1 |iL; lOX PCR buffer, 10 |iL; dNTP stock, 8 |aL, make up to 97.5 |aL with
distilled sterilized water
2. Perform the following annealing/extension step with the mix in step 1 using
a thermal cycler with the program: denaturation, 94°C for 30 s; cool to
37°C over a period of 30 min; maintain at 37°C over 15 min
3. Add 0.5 |iL Taq and mix gently. Spin down any droplets on the tube wall
and then incubate at 72°C for 3 mm.
4. To this reaction mix add: 1 )aL M13 primer M4 (20 pmol/|aL), 1 |j,L M13
primer RV (20 pmol/|aL).
5. Carry out PCR using the following program for 5—10 cycles: denaturation,
94°C for 30 s; primer annealing, 55°C for 2 mm; primer extension, 72°C
for 1 mm.
6. Remove an aliquot (5 |j,L) following the amplification reaction and con-
firm that a specific DNA product of the correct size has been generated.

*Different M13 primers must be used m a and b

PCR-Based Site-Directed Mutagenesis 163

7. Purify the remaining amplified DNA by phenol/chloroform extraction fol-

lowed by ethanol precipitation. Resuspend in 10 |nL distilled sterilized water.
3.3. Recloning of the Mutated Target DNA Sequence
The second round PCR product is then digested with two restriction
enzymes (X and Y in Fig. 1), which are dictated by the MUT primer/Ml3
primer combination used in the first round PCR reaction (see Note 2).
1. To 10 i^L of the purified second round PCR product, add 2 i^L of appropri-
ate lOX restriction buffer, 6 |aL of distilled sterilized water, mix, and then
add 1 i^L of each specified restriction enzyme. Mix gently and then incu-
bate at 37°C for at least 1 h.
2. Restrict the DNA of an appropriate cloning vector with identical enzymes.
Take 1 |j,g vector DNA, add 5 |u,L of appropriate lOX restriction buffer,
1 fiL of each restriction enzyme, and make up to an appropriate volume
with distilled water. Mix gently and incubate as before.
3. Purify the restricted PCR product and cloning vector by phenol/chloro-
form extraction and ethanol precipitation. Resuspend each purified DNA
in 2 |j,L TE or water.
4. Separate the restricted cloning vector by agarose gel electrophoresis.
Excise the desired band from the gel and recover the DNA using a
SUPREC-01filtercartridge. Purify the eluted DNA by phenol/chloroform
extraction and ethanol precipitation. Resuspend the DNA pellet m 20 (xL
TE or distilled sterilized water.
5. To 1 |aL of restricted cloning vector add 2 ^L PCR product (see Note 5),
1 U (cohesive end) or 100 U (blunt-end) T4 DNA ligase, 2 |iL lOX ligation
buffer, and then make up to 20 [iL with sterile distilled water. Mix gently
and incubate at 16°C for 16 h.
6. Transform 20 |j,L (maximum) of the ligation reaction into 100 |iL compe-
tent E coli cells using standard protocols.
Following transformation at least three clones should be submitted for
DNA sequencing to confirm that the desired mutation has been introduced.
An example of the utility of this PCR in vitro mutagenesis method can
be demonstrated by site-directed mutagenesis of codon 12 of the
protoncogene c-ki-ras, changing the usage from glycine to alanine
(G12A). The first stage involved cloning a 108 bp PCR fragment con-
taining c-ki ras codon 12 into the Hindi site of pUC 18. This was fol-
lowed by the first round PCR reactions using a combination of: a, Rl
primer (c-ki-ras/12 Ala type, "5'-GTTGGAGCTGCTGGCGTAGG-3"')
and M13-RV primer; b, MUTl primer (which flanks the Hindi cloning
164 Shimada

site) and M13-M4 primer. After the heteroduplex formation, the second
round PCR was performed with M l 3 primer M4 and M l 3 primer RV.
The resulting PCR fragment was purified and digested with EcoBJ and
HindlU. Only DNA fragments containing the c-ki-ras codon G12A
mutation were recloned, which was confirmed by DNA sequencing.

4. N o t e s
1. For the design of the mutagenic primer Rl, the following should be con-
sidered to ensure the oligonucleotide will anneal efficiently to the speci-
fied target: If a single- or double-point mutation is to be introduced, then
10 flanking bases should be incorporated into the primer sequence either
side of the mutation site. If the flanking regions are A + T rich or substan-
tial alterations are involved, this can be extended to 15 bases or more.
Ensure that the designed Rl primer does not have significant com-
plementarity to Itself or to its possible partners for PCR, particularly at
their 3' ends. This will avoid "primer dimer" formation in which two prim-
ers hybridize to each other forming a very effective substrate for PCR. If
possible keep the G + C composition of the primer to about 50-60% and
avoid long stretches of the same base.
2. The choice of the MUT primer used when designing the reaction will be
dependent on: the cloning site used for insertion of the target DNA
sequence (see Fig. 3); the direction of the Rl primer; the mutated fragment
must be recloned following mutagenesis, which should be undertaken
with unique restriction sites (enzymes X and Y in Fig. 1), i.e., that neither
of these enzymes should have sites within the cloned target DNA sequence,
a. For enzyme X (see Fig. 1), any of the restriction endonuclease recogni-
tion sequences mutated by the particular MUT primer used in the first
round PCR reaction (see Fig. 2) may be chosen. The choice of enzymes
that may be selected for by a particular MUT primer are:
MUT 1 EcoRl, Sad
MUT 2 Bamm,Xbal,Saa,Accl, Hindi
MUT 3 Sphl, Hindlll
MUT 4 Sphl, Hindlll
MUT 5 Bamm, Xbal, 5a/I, Accl, Hindi
MUT 6 EcoRl, Sad
h. For enzyme Y (see Fig. 1), any of the restriction enzymes with sites in
the multicloning site on the side flanking both the inserted DNA frag-
ment and the recognition sequence for enzyme X may be utilized.
3. MUT 2 and MUT 5 primers are sometimes difficult to anneal to the tem-
plate at 55°C. In such cases lower the annealing temperature to 45°C.
PCR-Based Site-Directed Mutagenesis 165

4. If any nonspecific bands appear on the agarose gel following electrophore-

sis of the first and second round PCR products, the desired band in
each case should be excised from the gel and purified. This may be
achieved by electroelution or by using commercial products such as
SUPREC-01 filter cartridges.
5. Recommended amounts of DNA are vector:insert = 0.03 pmol:0.1-0.3
pmol (0.03 pmol of pUC 18 DNA corresponds to about 50 ng).
References
1. Ito, W., Ishiguro, H., and Kurosawa,Y. (1991) A general method for introducing a
series of mutations into cloned DNA using the polymerase chain reaction. Gene
102, 67-70.
2. Hemsley, A., Amheim, N., Toney, M. D., Cortopassi, G., and Galas, D. J. (1989) A
simple method for site-directed mutagenesis using the polymerase chain reaction.
Nucleic Acids Res. 17, 6545-6551.
3. Higuchi, R., BCrummel, B., and Saiki, R. (1988) A general method of in vitro prepa-
ration and specific mutagenesis of DNA fragments, study of protein and DNA
interactions. Nucleic Acids Res. 16, 7351-7367.
4. Ho, S. N., Hunt, H. D., Horton, R. M., PuUen, J. K., and Pease, L. R. (1989) Site-
directed mutagenesis by overlap extension using the polymerase chain reaction.
Ge«e 77, 51-59.
5. Sambrook, J, Fritsch, E. P., and Maniatis, T. (1989) Molecular Cloning A Labo-
ratory Manual, 2nd ed , Cold Spring Harbor Press, Cold Spring Harbor, NY.