In Vitro Mutagenesis
M Arkin, Sunesis Pharmaceuticals, Redwood City, CA, USA
2001 Elsevier Inc. All rights reserved.
This article is reproduced from the previous edition, volume 2, pp 10101014, 2001, Elsevier Inc.
In vitro mutagenesis methods, especially site-directed muta
genesis, have revolutionized our understanding of protein
function and gene regulation. In vitro mutagenesis describes
the process by which a researcher alters one or more base pairs
in a cloned gene; expression of the gene yields a protein with
one or more altered amino acids. These mutant proteins may
show a change in function, such as lost or altered activity. The
ability to manipulate precisely the chemical nature of a gene
and therefore the protein encoded by this DNA has enabled
biologists to identify protein function, characterize protein
structure, and manipulate the activity of a protein in vivo.
Furthermore, protein engineers have used site-directed and
random mutagenesis procedures to create new proteins
designed to have unique or improved function.
In vitro mutagenesis has been enabled by a number of break
throughs in biotechnology. Other articles in this encyclopedia
describe discovery and uses of recombinant DNA, DNA poly
merases, the polymerase chain reaction (PCR), and restriction
endonucleases. This article will describe the application of
these technologies to the mutagenesis of recombinant genes.
Nonselective Mutagenesis
Deletions
Nested deletion mutagenesis has been used to identify func
tional domains of proteins and RNA. By this method, the
plasmid containing the gene of interest is linearized at a restric
tion site near the gene. The gene is then cleaved for discrete
amounts of time by the enzyme exonuclease III, which removes
bases from duplex DNA containing a 5 overhang. The result is
a nested set of plasmids in which the gene fragments vary in
length from one side of the gene and contain a common end.
These partially digested genes are then recloned into a plasmid
vector and transformed into Escherichia coli. In one early exam
ple of this method, researchers studying 5S ribosomal RNA
used exonuclease III to delete bases from the 5 end and identi
fied regions within the 5S rRNA gene which control its
transcription initiation.
Chemical Damage and Enzymatic Misincorporation
Chemical mutagenesis and enzymatic misincorporation tech
niques cause a small number of mutations throughout a piece
of DNA. Both methods yield a library of mutations that are
cloned into a plasmid and then screened or selected for func
tion. Commonly used chemicals include sodium bisulfite,
formic acid, and hydrazine. Sodium bisulfite causes the deami
nation of cytosine to uracil; during DNA synthesis, the altered
base is paired with adenosine instead of guanine. Hydrazine
and formic acid remove bases from the DNA strand, creating
abasic sites that can pair with any one of the four bases during
enzymatic synthesis. Nucleotides can also be altered at random
46 Brenners Encyclopedia of Genetics, 2nd Edition, Volume 4
sites through misincorporation of deoxyribonucleotide tripho
sphates (dNTPs) during DNA synthesis. For example, DNA
polymerase runs with impaired fidelity in the presence of man
ganese ions, and occasionally adds an incorrect base.
Alternatively, when one of the dNTPs is added in very low
concentrations, the enzyme will sometimes misincorporate
one of the other three bases. Certain dNTPs, such as
N6-hydroxydeoxycytidine, are also mutagenic and can cause
mispairing mutations. In all cases, the frequency of mutation
is increased by using DNA polymerase without a proofreading
function (such as Klenow fragment from E. coli). The modern
version of enzymatic misincorporation, error-prone PCR, is
regularly used to make mutant DNA libraries.
Site-Directed Mutagenesis
Site-directed mutagenesis involves the specific substitution of
one DNA base for another. Unlike the nonspecific mutations
described above, site-directed mutagenesis allows precise con
trol of the number, placement, and base substitution of
mutants. The two classes of site-directed mutagenesis include
methods that use double-stranded DNA cassettes and those
that use single-stranded oligonucleotide primers. All of the
techniques described here can give high yields of the desired
mutations; the choice of mutagenesis method is largely a mat
ter of convenience and personal preference.
Site-directed mutagenesis is possible because of the inven
tion of automated chemical synthesis of DNA and the
overexpression of DNA-processing enzymes. Through chemical
DNA synthesis, defined oligonucleotides up to 100 bases can
be prepared reproducibly and inexpensively. Synthetic oligo
nucleotides are used extensively for site-directed mutagenesis,
as primers for DNA polymerase and as oligonucleotide cas
settes. Equally important has been the identification and
overexpression of DNA-modifying enzymes, including restric
tion endonucleases for cleaving DNA at specific recognition
sites and DNA polymerases for generating double-stranded
DNA from a single-stranded template. Furthermore, the dis
covery of thermophilic DNA polymerases has enabled
PCR-based methods for site-directed mutagenesis.
Cassette Mutagenesis
In cassette mutagenesis, a synthetic double-stranded oligonu
cleotide cassette containing the desired mutations is docked
between two restriction enzyme sites on a plasmid vector. In
the simplest procedure, the restriction sites are separated by no
more than 100 base pairs; the ends of the oligonucleotide
duplex are complementary to the restriction cleavage sites so
that the cassette can be readily ligated into the plasmid
(Figure 1(a)). Since dozens of restriction enzymes are
doi:10.1016/B978-0-12-374984-0.00778-6
 In Vitro Mutagenesis 47

(a) (b)

dU a. Anneal mutagenic
oligo to dU-DNA
b. DNA polymerase

dU
BcgI c. DNA ligase

dU
dU

BcgI dU
dU
Transform into
dut + ung + strain
Cleave with

dU
of E. coli
BcgI

dU

Ligate cassette of any length

with complementary sticky ends

dU-DNA degraded; mutant propagated

Figure 1 Methods of oligonucleotide-directed mutagenesis. (a) Cassette mutagenesis with BcgI-containing plasmid. The BcgI-containing plasmid is
constructed by removing the region to be mutagenized and replacing it with a BcgI recognition sequence. Short arrows show sites of BcgI cleavage.
Following cleavage by the restriction enzyme, the mutagenic cassette is ligated into the gene. Note that the restriction sites are removed during
mutagenesis. (b) dU method for primer-based mutagenesis. A dU-containing single-stranded plasmid is prepared from an M13 vector in a dutung strain
of Escherichia coli. The mutagenic oligonucleotide is hybridized to the dU template (the mutagenic primer shown will create an insertion in the gene of
interest). The rest of the second strand is filled in by DNA polymerase and ligated by DNA ligase. Transformation into a du+ung+ strain of E. coli results in
degradation of the dU strand and propagation of the mutant.

commercially available, it is often possible to identify restric
tion sites near the sequence of interest.
One clever cassette design takes advantage of restriction
endonucleases such as BspMI and BcgI which cleave DNA
several base pairs away from their recognition sequences.
BcgI, for instance, cleaves DNA at any sequence 10 bases away
from each side of the enzymes specific binding site, while
BspMI cleaves on one side of an asymmetric recognition
sequence. The recognition sequence and product of BcgI clea
vage are shown below, where N is any nucleotide:
N10 CGA N6 TGC N12
oligonucleotide containing the desired mutation, called a
N12 GCT N6 ACG N10
mutagenic oligonucleotide, as a primer for DNA synthesis. By
To prepare a BcgI cassette, these recognition sequences are
added into a cloned gene by PCR such that the restriction site
replaces the region to be mutated (Figure 1(a)). A cassette is
synthesized to contain (1) the gene sequence that was removed
from the vector, (2) the desired mutations, and (3) ends com
plementary to the products of BcgI cleavage. The site-directed
mutant is then made by cutting the plasmid with BcgI and
ligating in the cassette. The advantage of these vectors is that
the restriction enzyme sites are cut out of the gene when the
cassette is added. Thus, the recombinant gene does not need to
contain unique restriction sites, and the wild-type vector can be
readily distinguished from the mutant by restriction digest.
Furthermore, since linear DNA is not readily transformed and
replicated in cells, precutting with the restriction enzyme before
transformation will increase the yield of mutant clones. This
type of cassette has been used in the mutational analysis of HIV
reverse transcriptase.
Primer-Directed Mutagenesis
General Methods
Site-directed mutagenesis can also be accomplished using an
this technique, the single-stranded oligonucleotide is
hybridized to a single-stranded plasmid, using bases
complementary to the wild-type gene. The mutagenic region
of the oligonucleotide can contain several single-base mis
matches, or it can be much longer or shorter than the
wild-type sequence (yielding insertions or deletions in the
mutated gene). DNA polymerase initiates synthesis of the
DNA at the oligonucleotide and fills in the second strand;
addition of DNA ligase seals the nick in the newly synthesized
strand. Transformation of this heteroduplex plasmid produces
both wild-type and mutant plasmids in E. coli, but several
methods (see below) have been devised to increase the propor
tion of mutants.
48 In Vitro Mutagenesis

DNA templates for oligonucleotide-based mutagenesis are
readily prepared using the single-stranded DNA bacteriophage
M13. Commercially available plasmids contain M13 replica
tion initiation sites as well as cloning sites with regulated
promoters. Thus, a single plasmid can be used for cloning,
M13 mutagenesis, and protein expression.
dU Method
Variations of the primer-based method increase the yield of
mutant by preferentially degrading the template strand. A com
monly used technique, first described by Kunkel, takes
advantage of dutung strains of E. coli (Figure 1(b)). Whereas
most bacteria will degrade DNA containing uracil (dU-DNA),
dutung strains are deficient in the degradation of both dUTP
(dut) and dU-DNA (ung). Thus, M13 templates isolated from
dutung bacteria will contain some dU in place of dT. After
hybridization of the mutagenic oligonucleotide, DNA synthesis
and ligation, the heteroduplex plasmid is transformed into a
dut+ung+ strain of E. coli which degrades the wild-type,
dU-containing template strand but not the newly synthesized
mutant strand. Thus, mostly mutagenic plasmid is propagated.
Other methods use similar approaches by adding methyl-dC or
thiophosphate-dC during in vitro DNA synthesis; these modifi
cations make the mutagenic strand resistant to degradation by
certain restriction enzymes.
Polymerase Chain Reaction
PCR-mediated mutagenesis is similar to the oligonucleotide
methods described above, in that a mutagenic oligonucleotide
is used as a primer for DNA synthesis. An advantage of the PCR
method lies in the inherent amplification of the mutagenic
DNA, which requires only a small amount of the wild-type
DNA as template. PCR mutagenesis can be performed on linear
pieces of DNA, such as restriction fragments, as well as on
circular plasmids. Figure 2 pictures some of the methods dis
cussed below.
PCR Mutagenesis of Linear DNA
If the desired mutation is found near a restriction enzyme site,
the mutation can be incorporated by preparing one PCR primer
containing the mutation and the restriction site and a second
primer containing a downstream restriction site. The PCR pro
duct is then treated with the restriction enzymes and ligated

(a) (b) (c)

2 4

4
2

1 3
1 3
PCR PCR
Two PCR reactions:
primers 1+ 2, 3 + 4

1 Phosphorylate Mix and

PCR with external and ligate anneal
primers

Gaps repaired in E. coli

Figure 2 Methods of PCR mutagenesis. (a) Extension-overlap PCR generates mutations between two restriction enzyme sites. Four primers are
prepared; two containing the restriction sites (primers 1 and 4) and two containing the mutagenic sequence (primers 2 and 3). After two PCRs, fragments
12 and 34 are combined and stitched together by PCR using primers 1 and 4. The long product 14 is restricted and ligated into the vector. (b) Inverted
PCR uses two back-to-back primers, one containing the mutations of interest. PCR yields the full-length, linear plasmid which is made into closed circular
DNA by DNA ligase. (c) Recombinant circle PCR uses two sets of primers. Primers 2 and 3 contain the mutagenic sites and prime opposite strands;
primers 1 and 4 prime from different positions on the plasmid. The PCR products 12 and 34 are truncated, linear versions of the plasmid; recombination
in vitro gives gapped plasmids which are repaired in E. coli.

into the plasmid as a DNA cassette. If there are no restriction
sites near the mutagenic sequence, overlap-extension PCR and
megaprimer PCR can be used to introduce the mutations.
Overlap-extension PCR requires four primers and three PCR
steps (Figure 2(a)). The first two PCR steps produce two
overlapping DNA fragments, both containing the desired
mutation. The final PCR step uses the outside primers to stitch
together the two fragments into the full-length cassette.
Megaprimer PCR, a variant of the overlap-extension method,
uses three primers and two PCR steps. The first step yields a
DNA fragment containing one restriction site and the muta
tions. This long DNA fragment is used as a megaprimer in the
second PCR step along with a primer containing the second
restriction site.
PCR Mutagenesis of Circular DNA
PCR mutagenesis can also be used to amplify the entire plas
mid containing the gene of interest. One straightforward
method, termed inverted or counter PCR, uses back-to-back
primers (Figure 2(b)); one PCR primer serves as the muta
genic oligonucleotide and the other oligonucleotide primes
from the opposite strand, adjacent to the mutagenic primer.
The PCR product is a full-length, linear plasmid which is then
phosphorylated and ligated before transformation. This
method can readily be used to make deletion mutants by
creating a gap between the primers. Variants of this method
include recombinant circle PCR (Figure 2(c)) and recombi
nation PCR, both of which rely on recombination of linear
plasmids. In these techniques, two inverse PCRs are per
formed with gapped primers at different sites. These two
mutant plasmids are then recombined in vitro by mixing and
annealing (recombinant circle PCR) or in vivo (recombination
PCR). The gaps are then repaired by the bacterial DNA repair
machinery.
Libraries of Mutations
Combinatorial and random mutagenesis methods create
libraries of DNA which are subsequently screened or selected
for function. By analyzing large numbers of clones simulta
neously, a small number of active mutants can be separated
from a pool of millions of variants. The preparation of libraries,
selection of winners and amplification of these selectants are
often called in vitro evolution.
Doped versus Saturation Mutagenesis
Libraries of mutant DNA molecules can be designed such that a
small number of random mutations are introduced throughout
the gene analogous to the nonselective mutagenesis described
above or large number of mutations are focused on a small
region of a gene. When all possible DNA mutations can be
found at a given site with equal frequency, the site is described
as saturated. When the number of mutations at a given site is
small, the site is said to be doped with the mutation.
Saturation mutagenesis is readily accomplished through auto
mated DNA synthesis. During synthesis, discrete bases are
added in sequence to the growing DNA chain; to saturate a
In Vitro Mutagenesis 49
position on this chain, equal amounts of all four bases are
added simultaneously. Doping can similarly be accomplished
by mixing a measured fraction of mutagenic base at a given site.
After these doped or saturated mutagenic oligonucleotides
have been synthesized, in vitro mutagenesis proceeds as usual
via cassette mutagenesis or primer-based mutagenesis.
Error-prone PCR offers an alternate method for doping
mutants throughout a gene; the rate of mutagenesis is ~0.7%
for Taq DNA polymerase. Both saturation and doping strategies
have been used to identify critical protein residues and to create
novel binding or catalytic functions. Examples include peptides
that antagonize or agonize cell-surface receptors, and enzymes
that are active in nonaqueous environments.
Mutagenesis and Recombination
An increasingly popular method for generating libraries of a
gene utilizes an in vitro recombination technique called
DNA shuffling. In DNA shuffling, one or more genes are
randomly chopped into smaller pieces of DNA by a nucle
ase and reconnected with a DNA polymerase. During this
reconstruction phase, homologous fragments of DNA can
anneal and prime each other, creating a recombined gene.
Mutations are incorporated into the genes via errors during
DNA polymerization. DNA shuffling has been used to opti
mize the function of proteins as well as the activity of
whole operons and viruses.
Prospects
In vitro mutagenesis has become an integral part of genetic
analysis. Controlled mutagenesis has identified the function
of new genes, a process termed reverse genetics, and allowed
dissection of the mechanism of known proteins. Additionally,
site-directed mutagenesis has become an important tool in
biotechnology. For example, the design of nonimmunogenic
antibodies for human therapeutics underscores the practical
benefits of mutagenesis and protein engineering. The availabil
ity of DNA-modifying enzymes, cloning vectors, and synthetic
DNA make site-directed mutagenesis straightforward in most
laboratories; its applications are limited only by the
imagination.
See also: DNA Sequencing; In V itro Evolution; Mutant Allele;
Mutational Analysis; Polymerase Chain Reaction; Recombinant
DNA; Restriction Endonuclease; Screening.
Further Reading
Boyer PL and Huges SH (1996) Site-directed mutagenic analysis of viral polymerases
and related proteins. In: Kuo LC, Olsen DB, and Carroll SS (eds.) Methods in
Enzymology, pp. 538555. San Diego, CA: Academic Press.
Chen K and Arnold FH (1993) Tuning the activity of an enzyme for unusual
environments. Proceedings of the National Academy of Sciences of the United States
of America, 90: 56185622.
Kunkel TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic
selection. Proceedings of the National Academy of Sciences of the United States of
America 82: 477492.
50 In Vitro Mutagenesis

Riechmann L, Clark M, Waldmann H, and Winter G (1988) Reshaping human antibodies
for therapy. Nature 332: 323327.
Smith M (1985) In vitro mutagenesis. Annual Review of Genetics 19: 423462.
Stemmer WPC (1994) DNA shuffling by random fragmentation and reassembly.
Proceedings of the National Academy of Sciences of the United States of America 91:
Tao BY and Lee KCP (1994) Mutagenesis by PCR. In: Griffin HG and Griffin AM (eds.)
PCR Technology, pp. 6983. Boca Raton, FL: CRC Press.
Watson JD, Gilman M, Witkowski J, and Zoller M (1992) Recombinant DNA, 2nd edn.,
San Francisco, CA: W. H. Freeman.

107147.