Genetics ii (site directed mutagenesis)

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SITE DIRECTED MUTAGENESIS


OUTLINE:



















SERIAL
NO.
CONTENTS PAGE NO.
1 Definition 2
2 Introduction 2
3 History 2
4 Method 3
5 Procedure 3
6 Approaches 6
7 Applications 8
8 References 9
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SITE-DIRECTED MUTAGENESIS
DEFINITION:
Site-directed mutagenesis (SDM) is a method to create specific, targeted changes in double
stranded plasmid DNA.
OR
Site-directed mutagenesis is a molecular biology method that is used to make specific and
intentional changes to the DNA sequence of a gene and any gene products. Also called site-
specific mutagenesis or oligonucleotide-directed mutagenesis, it is used for investigating the
structure and biological activity of DNA, RNA, and protein molecules, and for protein
engineering.
INTRODUCTION:
There are many reasons to make specific DNA alterations (insertions, deletions and
substitutions), including:
To study changes in protein activity that occurs as a result of the DNA manipulation.
To select or screen for mutations (at the DNA, RNA or protein level) that have a desired
property
To introduce or remove restriction endonuclease sites or tags
Site-directed mutagenesis is one of the most important techniques in laboratory for introducing
mutation into a DNA sequence. However, with decreasing costs of oligonucleotide
synthesis, artificial gene synthesis is now occasionally used as an alternative to site-directed
mutagenesis.
HISTORY
Early attempts at mutagenesis using radiation or chemical mutagens were non-site specific. Later
analogs of nucleotides and other chemicals (such as aminopurine, nitrosoguanidine and bisulfite)
were used to generate localized point mutations.
Site-directed mutagenesis was achieved in 1973 in the laboratory of Charles Weissmann using a
nucleotide analogue N
4
-hydroxycytidine, which induces transition of GC to AT. These methods
of mutagenesis, however, are limited by the kind of mutation they can achieve, and they are not
as specific as later site-directed mutagenesis methods.
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In 1971, Clyde Hutchison and Marshall Edgell showed that it is possible to produce mutants with
small fragments of phage X174 and restriction nucleases.
Hutchison later produced with his collaborator Michael Smith in 1978 a more flexible approach
to site-directed mutagenesis by using oligonucleotides in a primer extension method with DNA
polymerase.
For his part in the development of this process, Michael Smith later shared the Nobel Prize in
Chemistry in October 1993 with Kary B. Mullis, who invented polymerase chain reaction.
METHOD:
Formerly, a method pioneered by Kunkel (Kunkel, 1985) that takes advantage of a strain
deficient in dUTPase and uracil deglycosylase so that the recipient E. coli degrades the uracil-
containing wild-type DNA was widely used. Currently, there are a number of commercially
available kits that also require specific modification and/or unique E. coli strains (for example,
the Phusion Site-Directed Mutagenesis from Thermo and the GeneArt system from Life).
The most widely-used methods do not require any modifications or unique strains and
incorporate mutations into the plasmid by inverse PCR with standard primers. For these
methods, primers can be designed in either an overlapping or a back-to-back orientation.
Overlapping primer design results in a product that will re-circularize to form a doubly-nicked
plasmid. Despite the presence of these nicks, this circular product can be directly transformed
into E. coli, albeit at a lower efficiency than non-nicked plasmids. Back-to-back primer design
methods not only have the advantage of transforming non-nicked plasmids, but also allow
exponential amplification to generate significantly more of the desired product. In addition,
because the primers do not overlap each other, deletions sizes are only limited by the plasmid
and insertions are only limited by the constraints of modern primer synthesis. Currently, by
splitting the insertion between the two primers, insertions up to 100 bp can routinely be created
in one step using this method.
PROCEDURE:
Site-directed mutagenesis is an in vitro method for creating a specific mutation in a known
sequence, and is typically performed using PCR-based methods. Primers designed with
mutations can introduce small sequence changes, and primer extension or inverse PCR can be
used to achieve longer mutant regions. Using these site-directed mutagenesis techniques allows
researchers to investigate the impact of sequence changes or screen a variety of mutants to
determine the optimal sequence for addressing the question at hand. Here a simple method for
site-directed mutagenesis is described.
1. Primer design
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For every mutation, two oligonucleotides that anneal to complementary strands of the template
should be designed. The required mutation of interest is introduced at the 5' end of only one of
the primers by substitution of the nucleotide sequence. The oligonucleotides are located directly
adjacent to each other (on separate strands) in order to amplify the whole plasmid. In the case of
deletion mutagenesis, the primers are designed with a gap between them, corresponding to the
region to be deleted (Figure 1B). In the case of insertion, the additional nucleotide(s) are added at
the 5' end of one or both primers. One or both oligonucleotides in each reaction are
phosphorylated; this is required for ligation of the PCR products.
Oligonucleotides are either phosphorylated using Polynucleotide Kinase (PNK) (for
phosphorylation protocol, refer to Appendix) or phosphorylated by the oligo supplier.
Oligonucleotides may be HPLC or PAGE purified to ensure that they are full-length.
Further requirements concerning primer design includes having at least 17 nucleotides,
corresponding exactly to the DNA sequence and ending with G or C at the terminal 3' position.
Silent restriction sites (mutations which incorporate a restriction site without changing the amino
acid sequence) may also be included to facilitate analysis of the resulting clones. When
additional silent mutations are included in the primers, a minimum of 8 correct nucleotides
should be present between the silent mutation and the 3' end of the primers.

2. Traditional PCR
When PCR is used for site-directed mutagenesis, the primers are designed to include the desired
change, which could be base substitution, addition, or deletion.
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During PCR, the mutation is incorporated into the amplicon, replacing the original sequence.
Mutations introduced by PCR can only be incorporated into regions of sequence complementary
to the primers and not regions between the primers. Primers incorporating the desired base
changes are used in PCR. As the primers are extended, the mutation is created in the resulting
amplicon.


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Figure 1. Site-Directed Mutagenesis by Traditional PCR.

3. Primer Extension
Site-directed mutagenesis by primer extension involves incorporating mutagenic primers in
independent, nested PCRs before combining them in the final product. The reaction requires
flanking primers (A and D) complementary to the ends of the target sequence, and two internal
primers with complementary ends (B and C). These internal primers contain the desired mutation
and will hybridize to the region to be altered. During the first round of PCR, the AB and CD
fragments are created. These products are mixed for the second round of PCR using primers A
and D. The complementary ends of the products hybridize in this second PCR to create the final
product, AD, which contains the mutated internal sequence. Longer insertions can be
incorporated by using especially long primers, such as IDT Ultramer oligonucleotides.
To create a deletion, the internal primers, B and C, are positioned at either side of the region to
be deleted to prevent it from being incorporated within fragments AB and CD from the first
round of PCR. The complementary sequences at the ends of the these fragments, created by
primers B and C, enable hybridization of AB to CD during the second round of PCR, and the
final product with the desired deletion (AD) is created.

Figure 2. Site-Directed Mutagenesis by Primer Extension.
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(A) Insertion: Primers B and C contain the complementary sequence that will be inserted (blue
line). Two reactions are performed in the first round of PCR using primer pairs A/B (1) and C/D
(2). The resulting amplicons are mixed with primer pair A/D for the second round of PCR. The
complementary ends of the first round amplicons hybridize and the PCR creates the final product
with the desired insertion.
(B) Deletion: Primers B and C are located on either side of the sequence to be deleted, and
contain sequence from both sides of the deletion (black or gray additions that match the black or
gray original sequence). Two reactions are performed for the first round of PCR using primer
pairs A/B and C/D. The amplicons are mixed with primer pair A/D for the second round of PCR.
The overlapping regions of these amplicons hybridize and the PCR creates the final product with
the desired deletion.
3. Inverse PCR
Inverse PCR enables amplification of a region of unknown sequence using primers oriented in
the reverse direction. An adaptation of this method can be used to introduce mutations in
previously cloned sequences. Using primers incorporating the desired change, an entire circular
plasmid is amplified to delete , change or insert the desired sequence.

Figure 3. Site-Directed Mutagenesis by Inverse PCR.
The primers used are 5-phosphorylated to allow ligation of the amplicon ends after PCR. A high
fidelity DNA polymerase that creates blunt-ended products is used for the PCR to produce a
linearized fragment with the desired mutation, which is then recircularized by intramolecular
ligation. (A) Deletion: Primers that hybridize to regions on either side of the area to be deleted
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are used. (B) Substitution: One of the primers contains the desired mutation (blue bubble). (C)
Insertion: The primers hybridize to regions on either side of the location of the desired insertion
(black, dotted line). One primer contains the additional sequence that will be inserted (blue line).
APPROACHES TOWARDS MUTAGENESIS:
A large number of methods are available to affect site-directed mutagenesis,

although most of
them are now rarely used in laboratories since the early 2000s, as newer techniques allow for
simpler and easier ways of introducing site-specific mutation into genes.
a. Kunkel's method
In 1987, Thomas Kunkel introduced a technique that reduces the need to select for the mutants.
The DNA fragment to be mutated is inserted into a phagemid such as M13mp18/19 and is then
transformed into an E. coli strain deficient in two enzymes, dUTPase (dut) and uracil
deglycosidase (ung). Both enzymes are part of a DNA repair pathway that protects the bacterial
chromosome from mutations by the spontaneous deamination of dCTP to dUTP. The dUTPase
deficiency prevents the breakdown of dUTP, resulting in a high level of dUTP in the cell. The
uracil deglycosidase deficiency prevents the removal of uracil from newly synthesized DNA. As
the double-mutant E. coli replicates the phage DNA, its enzymatic machinery may, therefore,
misincorporate dUTP instead of dTTP, resulting in single-strand DNA that contains some uracils
(ssUDNA). The ssUDNA is extracted from the bacteriophage that is released into the medium,
and then used as template for mutagenesis. An oligonucleotide containing the desired mutation is
used for primer extension. The heteroduplex DNA that forms consists of one parental non-
mutated strand containing dUTP and a mutated strand containing dTTP. The DNA is then
transformed into an E. coli strain carrying the wildtype dut and ung genes. Here, the uracil-
containing parental DNA strand is degraded, so that nearly, the entire resulting DNA consists of
the mutated strand.
b. Cassette mutagenesis
Unlike other methods, cassette mutagenesis need not involve primer extension using DNA
polymerase. In this method, a fragment of DNA is synthesized, and then inserted into a
plasmid.
[13]
It involves the cleavage by a restriction enzyme at a site in the plasmid and
subsequent ligation of a pair of complementary oligonucleotides containing the mutation in the
gene of interest to the plasmid. Usually, the restriction enzymes that cut at the plasmid and the
oligonucleotide are the same, permitting sticky ends of the plasmid and insert to ligate to one
another. This method can generate mutants at close to 100% efficiency, but is limited by the
availability of suitable restriction sites flanking the site that is to be mutated.
c. PCR site-directed mutagenesis
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The limitation of restriction sites in cassette mutagenesis may be overcome using polymerase
chain reaction witholigonucleotide "primers", such that a larger fragment may be generated,
covering two convenient restriction sites. The exponential amplification in PCR produces a
fragment containing the desired mutation in sufficient quantity to be separate from the original,
unmutated plasmid by gel electrophoresis, which may then be inserted in the original context
using standard recombinant molecular biology techniques. There are many variations of the same
technique. The simplest method places the mutation site toward one of the ends of the fragment
whereby one of two oligonucleotides used for generating the fragment contains the mutation.
This involves a single step of PCR, but still has the inherent problem of requiring a suitable
restriction site near the mutation site unless a very long primer is used. Other variations,
therefore, employ three or four oligonucleotides, two of which may be non-mutagenic
oligonucleotides that cover two convenient restriction sites and generate a fragment that can be
digested and ligated into a plasmid, whereas the mutagenic oligonucleotide may be
complementary to a location within that fragment well away from any convenient restriction site.
These methods require multiple steps of PCR so that the final fragment to be ligated can contain
the desired mutation.
d. Whole plasmid mutagenesis
For plasmid manipulations, other site-directed mutagenesis techniques have been supplanted
largely by techniques that are highly efficient but relatively simple, easy to use, and
commercially available as a kit. An example of these techniques is the Quikchange
method, wherein a pair of complementary mutagenic primers are used to amplify the entire
plasmid in athermocycling reaction using a high-fidelity non-strand-displacing DNA polymerase
such as pfu polymerase. The reaction generates a nicked, circular DNA. The template DNA must
be eliminated by enzymatic digestion with a restriction enzyme such as DpnI, which is specific
for methylated DNA. All DNA produced from most Escherichia coli strains would be
methylated; the template plasmid that is biosynthesized in E. coli will, therefore, be digested,
while the mutated plasmid, which is generated in vitro and is therefore unmethylated, would be
left undigested. Note that, in these double-strand plasmid mutagenesis methods, while the
thermocycling reaction may be used, the DNA need not be exponentially amplified as in a PCR.
Instead, the amplification is linear, and it is therefore inaccurate to describe them as a PCR, since
there is no chain reaction.
Note that pfu polymerase can become strand-displacing at higher extension temperature (70C),
which can result in the failure of the experiment; therefore the extension reaction should be
performed at the recommended temperature of 68C. In some applications, this method has been
observed to lead to insertion of multiple copies of primers. A variation of this method, called
SPRINP, prevents this artifact and has been used in different types of site directed mutagenesis.
1. In vivo site-directed mutagenesis methods
2. Delitto perfetto
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3. Transplacement "pop-in pop-out".
4. Direct gene deletion and site-specific mutagenesis with PCR and one recyclable marker.
5. Direct gene deletion and site-specific mutagenesis with PCR and one recyclable marker
using long homologous regions.
6. In vivo site-directed mutagenesis with synthetic oligonucleotides.
APPLICATIONS:
Site-directed mutagenesis is used to generate mutations that may produce rationally
designed protein that has improved or special properties.
Investigative tools - specific mutations in DNA allow the function and properties of a DNA
sequence or a protein to be investigated in a rational approach.
Commercial applications - Proteins may be engineered to produce mutant forms that are
tailored for a specific application. For example, commonly used laundry detergents may
contain subtilisin, whose wild-type form has a methionine that can be oxidized by bleach,
significantly reducing the activity the protein in the process. This methionine may be replaced by
alanine or other residues, making it resistant to oxidation thereby keeping the protein active in
the presence of bleach.
REFERENCES:
1. Zoller MJ (1991) New molecular biology methods for protein engineering. Curr Opin
Biotechnol, 2(4): 526531.
2. Reikofski J and Tao BY (1992) Polymerase chain reaction (PCR) techniques for site-directed
mutagenesis. Biotechnol Adv, 10(4): 535547.
3. Ho SN, Hunt HD, Horton RM, et al. (1989) Site-directed mutagenesis by overlap extension
using the polymerase chain reaction. Gene, 77(1):5159.
4. Shortle, D.; Dimaio, D.; Nathans, D. (1981). "Directed Mutagenesis". Annual Review of
Genetics 15: 265294.
5. Flavell R A, Sabo D L, Bandle E F, and Weissmann C (1975)."Site-directed mutagenesis:
effect of an extracistronic mutation on the in vitro propagation of bacteriophage Qbeta
RNA".Proc Natl Acad Sci U S A. 72 (1): 367371.s