Professional Documents
Culture Documents
Contributors
Adam Clore, PhD, Brian Reinertson,
and Scott Rose, PhD
WWW.IDTDNA.COM
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.1 Site-directed Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.1.1 PCR for Substitutions, Additions, and Deletions . . . . . . . . . . . . . 7
1.1.2 Primer Extension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.1.3 Inverse PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.1.4 Cassette Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.2 Random Mutagenesis (in vitro saturation) . . . . . . . . . . . . . . . . . . . . 16
1.2.1 Error-prone PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
1.2.2 PCR with Degenerate Primers . . . . . . . . . . . . . . . . . . . . . . . 19
1.2.3 Chemical Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2. Mutagenesis with Ultramer Oligonucleotides . . . . . . . . . . . . . . . . . . 23
2.1 Ultramer Primer Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.1.1 Terminal Changes by PCR . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.1.2 Terminal Additions by PCR . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.1.3 Oligonucleotide-directed Internal Mutagenesis . . . . . . . . . . . . 28
2.2 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.2.1 PCR Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.2.2 Ligation Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.2.3 DpnI Digestion Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.2.4 Transformation Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.3 Example Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.3.1 Protocol for Terminal Changes or Additions . . . . . . . . . . . . . . . 31
2.3.2 Protocol for Oligonucleotide-directed Internal Mutagenesis . . . . 35
3. Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.1 Primer Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3.1.1 Good Primer Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3.2 Template Concentration and Quality . . . . . . . . . . . . . . . . . . . . . . . 40
3.2.1 Too Much or Too Little Template . . . . . . . . . . . . . . . . . . . . . . 40
3.2.2 Poor Quality Template . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
2
3.3 Reaction Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
3.3.1 Experimental Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
3.3.2 Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
3.3.3 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
3.4 Reaction Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
3.4.1 Polymerases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
3.5 PCR Reaction Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.5.1 Cycle Number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.5.2 Annealing Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.5.3 Extension Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.5.4 Denaturation Temperature . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.5.5 Initial Denaturation Time . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.5.6 Touchdown PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.6 Ligation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.6.1 Quantification of Product . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.6.2 Gel Confirmation of a Ligation Reaction . . . . . . . . . . . . . . . . . 45
3.6.3 Inhibitors of Ligase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
3.7 DpnI Digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.7.1 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.7.2 Methylated DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.8 Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.8.1 Handling Competent Cells . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.8.2 Heat Shock Considerations . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.8.3 Electroporation Considerations . . . . . . . . . . . . . . . . . . . . . . 48
3.8.4 Antibiotic Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.8.5 Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.8.6 E. coli Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.8.7 Toxic Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
4. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
3
Mutagenesis Application Guide
1. Introduction
In vitro mutagenesis is used to purposefully change genetic information. Analysis of the
subsequent changes in gene expression and gene products helps elucidate the func-
tional effect of the mutation [1]. The technique falls into two general categories: site-
directed mutagenesis and random mutagenesis. Each contains several subcategories
with various methodologies used to generate mutations.
The particular mutagenesis method you choose to use will depend on the goal of the
project and the information you have about the target sequence (Table 1). Site-directed
mutagenesis creates a specific change in a known sequence, while random mutagen-
esis allows researchers to screen for mutations regardless of the genomic location or
to quickly create a wide variety of individual mutations. For both methods, a successful
experiment depends on many factors including the technique used for constructing
the mutant DNA, the quality of the primers used, an appropriate expression vector and
system, effective purification, and development of an assay for detection [2].
6
Site-directed mutagenesis is typically performed using PCR. The identification and con-
struction of new commercial polymerases and advances in oligonucleotide synthesis
have dramatically increased its efficiency. Primers designed with mutations can intro-
duce small sequence changes, and primer extension or inverse PCR can be used to
achieve longer mutant regions. In addition, PCR has provided increased precision along
with a decrease in cost and time spent on mutagenesis experiments [3]. While the rest of
this section will discuss the various PCR methods for site-directed mutagenesis, Section
2 will show how some of these mutation strategies can be more easily accomplished
using long oligonucleotides called Ultramer Oligonucleotides.
While PCR for substitutions, additions, and deletions is a simple way to introduce a muta-
tion, it is limited by the fact that the mutation can only be introduced in the sequence
covered by the primers rather than the sequence that lies between the primers [3].
7
Mutagenesis Application Guide
Existing sequence
5ggacgcaagctgtaatgctctagacgtta-------------tgtggtcattgtgtcaccgcaggacattga 3
3cctgcgttcgacattacgagatctgcaat-------------acaccagtaacacagtggcgtcctgtaact 5
Desired sequence
5ggaTCcaagctgtaatgctctagacgtta-------------tgtggtcattgtgtcaccgcaggacattga 3
3cctAGgttcgacattacgagatctgcaat-------------acaccagtaacacagtggcgtcctgtaact 5
Primer A
5ggaTCcaagctgtaatgctcta
||| |||||||||||||||||
3cctgcgttcgacattacgagatctgcaat-------------acaccagtaacacagtggcgtcctgtaact 5
5ggacgcaagctgtaatgctctagacgtta-------------tgtggtcattgtgtcaccgcaggacattga 3
||||||||||||||||||
<-cagtggcgtcctgtaact 5
Primer B
Figure 1A. PCR for Base Substitutions. Primers containing the base changes of interest as a non-comple-
mentary break in the primer sequence (indicated by the blue bubble in primer A) are used in a PCR reac-
tion. As the primers are extended, the resulting amplification product incorporates the mutation, replac-
ing the original sequence (shown as a blue bar in the PCR product).
8
Figure 1B. PCR for Deletions. Primer A contains complementary sequence to the regions flanking the area to
be deleted. During PCR, primer binding will cause a region of the template to loop out, and amplify only the
complementary region. The final product is shorter because it is missing the deleted sequence.
Figure 1C. PCR for Terminal Additions. An Ultramer primer containing an addition to the sequence on the
5 end (the 6X His tag, primer B) is used along with the complementary primer A to amplify a new product
containing the terminal addition.
9
Mutagenesis Application Guide
Primer extension for additions requires that any additional bases must reside within one
of the primers. Traditionally, primer length was limited to 3050 bases due to synthe-
sis yield constraints. However, with the advent of improved synthesis technology, use of
especially long primers, such as the IDT Ultramer Oligonucleotides, now makes longer
additions possible.
10
Figure 2B. Primer Extension for Deletions. Prim-
ers B and C are located on either side of the se-
quence to be deleted and contain sequence from
both sides of the deletion (indicated by black or
gray additions that match the black or gray origi-
nal sequence). This sequence will allow them to
overlap with the other fragment after the first
round of PCR. The first round of PCR uses primer
pairs A/B and C/D. The two resulting PCR products
are mixed together with the primer pair A/D for
a second round of PCR. The overlapping regions
of these two, first-round PCR products allow the
strands to hybridize and the second round of PCR
creates the final, full-length product with the de-
sired area deleted.
Alternatively, Lee t al. recently described a new method, overlap extension PCR, that in-
volves an additional set of PCR primers to bring in a longer insert [7]. Primer sets A/B and
E/F are used to amplify the original sequence while primer set C/D is used to amplify the
insert cassette. Primers B, C, D, and E have additional overlapping sequence that, when
hybridized, align the three sections in the correct order. Thus, during a second round of
PCR using the primer set A/F, the three separate pieces are incorporated together (Figure
2C).
11
Mutagenesis Application Guide
One of the most widely adopted methods for introducing changes by primer extension
was developed by Stratagene and is marketed as the QuikChange Site-Directed Muta-
genesis Kit. This mutagenesis protocol requires two complementary oligonucleotides, a
high-fidelity polymerase, and the restriction enzyme, DpnI. The approach is to hybridize
complementary oligonucleotides that contain altered sequence at their center to de-
natured double-stranded plasmid DNA. A high-fidelity polymerase is used to generate
a copy of each strand of the plasmid DNA by priming from the mutagenic primers. This
polymerase does not displace the newly synthesized strands and so the extension stops
when the primers copy the entire plasmid and return to the 5 end of the primer. The
extension mix is treated with the DNA endonuclease restriction enzyme, DpnI, which
requires that the N7 position of adenine be methylated as part of its GMATC recognition
sequence. The methylated adenine is only present on the parental plasmid (due to the
action of the bacterial DNA methyltransferase, Dam). Thus, DpnI selectively cleaves the
parental plasmid DNA, leaving only the mutagenic strands. Once transformed into high
efficiency competent bacteria, the annealed mutagenic strand nicks are sealed and the
plasmid, now carrying the mutation, is replicated.
An updated version of this method with a protocol for its use was recently published
by Erster and Liscovitch [10]. The authors suggest this method could be used for many
types of insertions into target proteins including: ligand-binding domains, functional
domains, cleavage sites, tags, and regulatory elements.
12
Figure 3A. Inverse PCR for a Deletion. This method uses primers that hybridize
to regions on either side of the area to be deleted. In this case, the primers
contain 5 phosphorylated ends to allow the two ends to be ligated together
following amplification. PCR with a high fidelity DNA polymerase that leaves
blunt ends creates a linearized fragment that is missing the deleted region.
This fragment is then recircularized by intramolecular ligation and the result-
ing plasmid is transformed.
13
Mutagenesis Application Guide
Figure 3B. Inverse PCR for a Substitution. One of the two primers contains the
mutation of interest (indicated by the blue bubble). In this case, both primers
contain 5 phosphorylated ends to allow the two ends to be ligated together
following amplification. PCR is used to amplify the entire circular plasmid to
create a linear template that contains the substituted sequence. This fragment
is then recircularized by intramolecular ligation and the resulting plasmid is
transformed.
14
Figure 3C. Inverse PCR for an Insertion. The primers are lined up back-to-back
on either side of the area where the new sequence will be inserted (indicat-
ed by the black, dotted line). One of the primers contains the additional se-
quence that will be inserted (indicated by the blue line). Both primers contain
5 phosphorylated ends to facilitate ligation following amplification. PCR cre-
ates a linearized fragment containing the new sequence. The plasmid is then
recircularized by intramolecular ligation and transformed.
15
Mutagenesis Application Guide
Cassette mutagenesis has many advantages. It is a very efficient technique and provides
an easy way to screen mutants by simply sequencing the resulting plasmids. It provides
flexibility to perform many different mutagenesis events on the same vector once set
up with flanking restriction sites. Multiple cassettes can also be inserted throughout the
vector [12]. Disadvantages to this approach include the need to identify or insert appro-
priate restriction sites for exchanging cassettes. Unless the mutated cassette is obtained
commercially, the mutations must be generated using oligonucleotides, cloned, and se-
quenced before insertion back into the plasmid. Finally, cassette mutagenesis can lead to
occasional double mutants or deletion mutants resulting from oligonucleotide impurities,
underscoring the importance of using high quality primers. (See Oligonucleotide Quality
Requirements for Mutagenesis Protocols, page 22, for more information on the impor-
tance of oligonucleotide purity in mutagenesis).
16
Figure 4. Cassette Mutagenesis. The original plasmid is cleaved with restric-
tion enzymes A and E on either side of the cassette to be removed (indicated
in orange). The restriction digest creates a linearized plasmid fragment and a
cassette. The new cassette (indicated in blue) containing the desired changes
is then ligated into the linearized plasmid.
17
Mutagenesis Application Guide
The area that undergoes mutagenesis in error-prone PCR is determined by the position
of the primers. The actual reaction is performed under conditions that reduce the fidel-
ity, or increase the error rate, of the polymerase. Thus, the number of reaction cycles
determines the degree of mutagenesis [20].
18
1.2.2 PCR with Degenerate Primers
PCR with degenerate primers can also be used for semi-random mutagenesis. In this
approach, primers are synthesized with a mixture of wild type and non-wild type nu-
cleotides which results in predictable rates of misincorporation per nucleotide [22]. The
primers can also contain both regions of wild type sequence and degenerate sequence.
Degenerate regions are synthesized with a mixture of the nucleotides consisting primar-
ily of the wild type base with a small percentage of the other three bases. Oligonucle-
otides with mixed base composition can be ordered from commercial vendors like IDT.
The degenerate PCR method is the most cost-effective method for performing satura-
tion mutagenesisgeneration of all possible mutations within a gene regiondue to
the shorter oligonucleotides used [22]. In addition, degenerate PCR also biases against
mutants with multiple mutations when there is a single degenerate base position. This
makes it a good tool for identifying single base changes that alter function. Because the
percentage of base substitutions at each position in the primer can be controlled, this
method allows for more precise control over the location and rate of mutagenesis than
other random mutation methods. The disadvantages to this method are that it can bias
mutations toward sequences with a higher binding affinity for the degenerate primers
and changes are limited to the primer binding locations. In addition, it is often hard or
impossible to select for changes that will result in a subset of amino acids.
19
Mutagenesis Application Guide
20
IDT Product Focus: Degenerate Primers
Machine Mixed Bases
Machine mixed bases contain an equal ratio of each base and are used to create
random primers. To order, enter the IUB symbols (e.g., R for a mix of A and G) into
the sequence on the IDT DNA ordering page (www.idtdna.com/order). For a com-
plete list of the IUB symbols, see the Mixed Bases tab on the DNA ordering page.
Custom Mixed Bases
Custom mixes of bases allow customers to specify the ratio of each base. Both
hand mix and machine mix options are available. To order click on the Mixed Bases
tab on the IDT DNA ordering page (www.idtdna.com/order).
Trimers
Inserting serial N bases gives rise to all 64 possible codons. This does not pro-
duce an equal representation of the 20 amino acids (AAs), but rather biases toward
those AAs that have more codons encoding them. Additionally, serial N bases will
also insert unwanted stop codons. To avoid this, a set of trimer phosphoramidites
have been developed which comprise a single codon for each of the 20 AAs. These
are available as a 20 Trimer Mix for creating better N-domains in oligonucleotides
intended to encode proteins. It is also possible to obtain custom mixes with more
limited amino acid content. To add Trimers to your oligonucleotide order, click on
the modification tab on the IDT DNA ordering page (www.idtdna.com/order).
Universal bases
To add these modifications to your oligonucleotide order, click on the modification
tab on the IDT DNA ordering page (www.idtdna.com/order).
5-Nitroindole is currently the best universal base available. It does not favor
any particular base pairing and is not as destabilizing to the duplex as mis-
matches between the standard bases. 5-Nitroindole directs random incorpo-
ration of any specific base when used as a template for DNA polymerase, and
partially blocks enzyme processivity.
21
Mutagenesis Application Guide
In addition to purity, IDT tests its oligonucleotides against those from competitors
in functional studies. A recent performance test examined primers used for site-
directed mutagenesis (SDM). Four pairs of SDM primers were ordered from four dif-
ferent companies (including IDT). These sets were:
These oligonucleotides were used in parallel SDM experiments, and resulting clones
were screened by IDT scientists. The data from the cumulative cloning experiments
show that, in every case, use of IDT oligonucleotides led to better mutagenesis results.
22
2. Mutagenesis with Ultramer Oligonucleotides
Ultramer primers are oligonucleotides that range in length from 25200 bases and sim-
plify the addition of large changes into a target sequence. These high-fidelity oligonu-
cleotides are the longest oligonucleotides commercially available. Coupling efficiencies
for their synthesis routinely reach 99.7%, which eliminates the need for purification.
Ultramer primers are especially useful in PCR and site-directed mutagenesis reactions.
Incorporating Ultramer Oligonucleotides as the mutagenic primers allows for greater
flexibility in the type and size of sequence changes that can be made. The 3 end of the
Ultramer Oligonucleotide primes the PCR with the additional sequence placed upstream
(5). Researchers can add long stretches of new sequences to an existing clone by adding
up to 180 bases to a 20 base PCR primer, make changes to a large area within a clone
in a single reaction, or change or correct multiple sequence locations simultaneously
(Figure 5). Ultramer primers also provide a new tool for regions that have traditionally
been difficult to target by extending primers into regions with more optimal sequence
composition than was previously attainable.
Changing sequence identity at the 5 and 3 ends of a known sequence, or adding ad-
ditional flanking sequences, are the most straightforward ways to modify DNA. Typically,
this procedure is used for adding terminal restriction sites to aid in cloning, adding pro-
tein purification tags such as 6X His, or adding bacterial phage promoters such as T7/T3/
SP6 for synthesis of in vitro RNA transcripts. However, site-directed mutagenesis makes it
possible to introduce sequence changes anywhere within a cloned sequence. Here we
provide design recommendations for using Ultramer primers with these types of muta-
genesis procedures as well as example protocols. The first techniques, Terminal Changes
by PCR and Terminal Additions by PCR, create changes at the ends of a PCR amplicon.
The second approach, Oligonucleotide-directed Internal Mutagenesis, creates changes
anywhere within a cloned sequence.
23
Mutagenesis Application Guide
mutations outside the 33-base region, almost all only contain mutations in the target-
ed area. This experiment exhibits both how Ultramer Oligonucleotides can be used for
creating multiple mutations in a broad region and how additional purification of these
products is not mandatory.
Figure 5A. 93mer Ultramer Primers Used for Mutagenesis (Above left) Aspartic acid residue 7 and glutamic
acid residue 8, contribute to the active site of the Pyrococcus abysii RNase H2 enzyme. Targeted saturation site-
directed mutagenesis was carried out on residues 212 using a 93mer Ultramer Oligonucleotide. 2 desalted
complementary Ultramer Oligonucleotides were synthesized with 30 base non-degenerate 5 and 3 ends. The
internal sequences of the Ultramer Oligonucleotides were synthesized with mixed phosphoramidites at a ratio
of 91:3:3:3 so that mutations would be introduced but not at every site. 2 desalted 93mer degenerate Ultramer
Oligonucleotides were run on a 10% acrylamide, 7 M Urea denaturing gel and visualized with GelStar Nucleic
Acid Gel Stain (Cambrex Bio Science Rockland). The size markers were PAGE purified Ultramer Oligonucleotides
of 80, 100, 125, 150, 175, 200, and 225 nt in length.
Figure 5B. Sequence of RNase H2 Enzyme Active Site Mutations (Above right) The mutagenesis reaction was
completed by extension with KOD DNA polymerase, followed by DpnI digestion of the parent plasmid, and
transformation into competent BL21DE3 cells. 59 of the resultant clones were sequenced. Even though the
Ultramer Oligonucleotides were not purified further than standard desalting, 88% of the clones contained
only mutations within the central 11 amino acid targeted region. The majority of these clones had between 35
mutations within this region.
24
2.1 Ultramer Primer Design
Primer design and cycling conditions will vary based on the type of sequence change
you intend to make. While some of the basic PCR primer design rules hold true, others
may have to be amended as the template sequence places some constraints on the
primer sequence.
The primer sequence should have little to no secondary structure. You can measure the
stability of any secondary structure within the oligonucleotide sequence with UNAfold,
another free tool within the online suite of IDT SciTools (www.idtdna.com/SciTools). In
the output, confirm that the Tm of the folded sequence is at least 510C less than the
annealing temperature and the secondary structure has a G value between 0 and -9 kcal/
mol. For additional discussion, see Troubleshooting Section 3.1.1 Good Primer Design.
25
Mutagenesis Application Guide
Example: Changing a base near the 5 end of a sequence to generate a Bam HI (GGATCC)
restriction site.
The Tm was calculated using OligoAnalyzer 3.1 with the following conditions: 0.25 M
oligo, 50 mM KCl, 2 mM MgCl2, 0.8 mM dNTP. Cycling conditions used with the hot start
KOD DNA Polymerase (0.5 U) (Novagen) were 2 min 95C; 30 x (20 sec 95C, 15 sec 56C,
45 sec 70C).
If the PCR product will be digested with a restriction enzyme and subcloned directly,
add 35 T bases at the 5 end of the primer to allow for efficient binding and cleavage
of the PCR product by the restriction enzyme. Do not include those additional bases in
the Tm calculation.
26
2.1.2 Terminal Additions by PCR
See Section 2.3.1 for an example protocol for this method.
To design the primers, select the sequence by extending the length base-by-base from
the 5 end of each DNA strand until the calculated Tm of the proposed primer sequence
matches the desired annealing temperature for the PCR reaction, typically in the range
of 5865C. The oligonucleotide concentration, monovalent salt concentration, dNTP
concentration, and Mg2+ ion concentration are necessary for accurate Tm calculations.
Exclude primer designs that form stable heterodimers, homodimers, or hairpins, if pos-
sible. While it may not be possible to completely eliminate such undesired interactions,
increasing or decreasing primer length should minimize these interactions. Potential
interactions with heterodimers, homodimers, and hairpins can be evaluated using Oli-
goAnalyzer 3.1 (www.idtdna.com/OligoAnalyzer). Once the forward and reverse primers
have been designed, add the new sequence in front of the appropriate primer. The PCR
thermocycling profile is based on the initial annealing temperature of the primer with-
out the additional sequence added.
Example: Adding HSV-Tag, 6X His tag to the 3 end of the coding sequence for the Pyro-
coccus abysii RNase H2 gene.
Target sequence:
atgaaagttgcaggtgcagatgaagctggtcgtggtccagttattggtccgctggttattgttgctgctgttgtggag-
gaagacaaaatccgctctctgactaagctgggtgttaaagactccaaacagctgaccccggcgcaacgtgaaaaactgttc-
gatgaaatcgtaaaagtactggatgattactctgtggtcattgtgtccccgcaggacattgacggtcgtaagggcagcat-
gaacgaactggaggtagaaaacttcgttaaagccctgaatagcctgaaagttaagccggaagttatttacattgattc-
cgctgatgttaaagctgaacgtttcgctgaaaacattcgcagccgtctggcgtacgaagcgaaagttgtagccgaacata-
aagcggatgcgaagtatgagatcgtatccgcagcctctatcctggcaaaagttatccgtgaccgcgagatcgaaaagct-
gaaagccgaatacggtgattttggttccggttacccgtctgatccgcgtactaagaaatggctggaagaatggtatag-
caaacacggcaatttcccgccgatcgtgcgtcgtacttgggatactgcaaagaaaatcgaagaaaaattcaaacgtgcg-
cagctgaccctggacaacttcctgaagcgttttcgcaac
The following primers were selected. Tm was calculated using OligoAnalyzer 3.1 with the
following conditions: 0.25 M oligo, 50 mM KCl, 2 mM MgCl2, 0.8 mM dNTP.
Forward primer
5-atgaaagttgcaggtgcaga-3 Tm 61.6
Reverse primer
5-gttgcgaaaacgcttcagga-3 Tm 62.6
Extended reverse primer with HSV-Tag (in green) and 6X His tag (in red)
5-TCAGTGGTGGTGGTGGTGGTGCTCGACATCCTCGGGGTCTTCCGGGGCGAGTTCTGGCTGGCTgttgcgaaaa
cgcttcagga-3
27
Mutagenesis Application Guide
As discussed in Section 1.1.2, one of the most widely adopted methods for introducing
changes anywhere within a plasmid was developed by Stratagene and marketed as the
QuikChange Site-Directed Mutagenesis Kit. Incorporating Ultramer primers into this
method is both simple and highly effective.
The Ultramer primer sequence is designed by selecting 25 bases upstream and down-
stream (shown underlined, below) of the site to be mutagenized (shown in red, below).
The goal is to get the Tm of this flanking sequence to be 60C or greater. Add in the
desired sequence (shown in blue, below) to finalize the Ultramer primer sequence, and
create the reverse complement sequence. The Stratagene protocol recommends puri-
fied primers but this step is not necessary when high-fidelity Ultramer Oligonucleotides
are useddesalted oligonucleotides of this length do not require additional purification
as long as more than one clone will be sequenced.
5 GGATCCGATGAAAGTTGCAGGTGCAAGGAAGGCTGGTCGTGGTCCAGTTATTGGTC 3
5 GACCAATAACTGGACCACGACCAGCCTTCCTTGCACCTGCAACTTTCATCGGATCC 3
28
2.2 Controls
Effective controls are an integral component of any mutagenesis experiment. The con-
trols listed below will confirm each step in the process toward making a site-specific mu-
tation. You may choose to eliminate some of these controls but it is important to weigh
removing a control against the time and costs associated with repeating the experiment
if you observe incomplete or poor results.
At a minimum, each step of the process should include a positive and a negative control.
The positive control should be a plasmid of similar size to the experimental plasmid that
can be mutated to give an easily identifiable phenotype. This will allow you to follow
each of the steps of the mutagenesis reaction with template and primers that are known
to work. An example of this type of control would be a plasmid containing a gene ex-
pressing the alpha subunit of the -galactosidase gene. This gene can easily be mutated
into an inactive form by changing the start codon to a stop codon, or by adding one or
more stop codons to the 5 end of the gene. Detection of this change is easily observed
when transformed into cells compatible with blue-white screening (such as DH5) and
plated on agar plates containing X-Gal.
Positive controla PCR containing a control plasmid and primers that will yield
a mutant product. This reaction should be run with the same conditions and
reaction components as the experimental reaction. The control plasmid should
be similar in size to the test sequence so that it will amplify under the same
cycling conditions.
When you run these controls on an agarose gel, you should see a strong, single
band in the positive PCR control lane and no band in the negative control lane.
29
Mutagenesis Application Guide
A lack of colonies from any of the controls indicates a problem with the competent cells,
the transformation protocol, or the media used to select for the correct transformants. If
this is the case, see Troubleshooting Section 3.8 Transformation.
30
2.3 Example Protocols
In this section, we provide two protocols: one for Terminal Changes or Additions and one
for Oligonucleotide-directed Internal Mutagenesis. We also provide reaction setup sug-
gestions for two different types of polymerases: Phusion (Thermo Fisher, New England
Biolabs) and KOD (Novagen). These are general protocols and may need to be altered
depending on your specific application.
Protocol for Terminal Changes or Additions see Section 2.3.1, page 31.
Protocol for Oligonucleotide-directed Internal Mutagenesis see Section 2.3.2, page 35.
1. PCR
Set up a 50 L mutagenesis reaction using a high fidelity polymerase, such as Phusion
(Thermo Fisher, New England Biolabs) or KOD DNA polymerase (Novagen). A typical re-
action setup and cycling times are described below.
31
Mutagenesis Application Guide
PCR Cycling Parameters for KOD DNA Polymerase PCR Cycling Parameters for Phusion DNA Polymerase
Reactions that do not produce a single clean band on a gel may require either gel puri-
fication or PCR optimization.
3. Ligation
Circularize the amplicon with T4 DNA Ligase.
This reaction can be carried out at room temperature (22C) for 5 min if the Quick Liga-
tion Kit (New England Biolabs) is used. Alternatively, a standard T4 DNA Ligase can
be used; follow the manufacturers instructions. As Taq and other thermostable DNA
ligases do not efficiently ligate blunt ended DNA, we do not recommend their use for
this application.
PCR product 9 L
2X Quick T4 DNA Ligase Buffer (NEB) 10 L
T4 DNA Ligase (NEB) 1 L
4. DpnI Digestion
Add 1 L of DpnI restriction endonuclease (New England Biolabs) to the reaction mix-
ture to remove the original plasmid DNA. Incubate at 37C for 30 min.
32
5. Transformation
Transform 2 L of the final product into competent E. coli following the manufacturers
instructions. For DNA fragments smaller than 12 Kb, chemically competent cells such as
DH5 can be purchased or prepared in the lab. Larger plasmids require electroporation
for efficient uptake by bacteria.
Typical transformations use 25100 L of competent cells under the following conditions:
1. Place the competent cells on wet ice until completely thawed (25 min).
2. Add 25 L of the DpnI digestion product, not exceeding 10% of the competent
cell volume.
3. Incubate on wet ice for 30 min, stirring gently every 10 min. Do not pipette or vortex
cells.
4. Place the cells in a 42C water bath for 3045 sec.
5. Immediately return the cells to wet ice for 2 min.
6. Add 10 volumes of SOC (e.g., for 25 L cells, add 250 L SOC) and place in a shaking
incubator at 37C for 1 hr. See below for an SOC media recipe.
7. Plate 100200 L of the reaction on 10 cm LB agar plates with the appropriate selec-
tion agent (e.g., 100 g/mL ampicillin or 50 g/mL kanamycin).
8. Place in an incubator at 37C overnight.
33
Mutagenesis Application Guide
6. Screening
Methods of screening for desired mutations vary depending on the size and complexity
of the mutated area. In general, Sanger sequencing using Applied Biosystems BigDye
Terminators and capillary sequencing is the preferred method. A protocol for a rapid pu-
rification of DNA to screen colonies is listed below. Alternatively, a plasmid purification
kit such as Qiagens Plasmid Mini Kit can be used. Screen 520 colonies to ensure identi-
fication of a correct clone. Note that primers used for sequencing should be positioned
at least 40 bases away from the mutation site as the first few bases of sequencing reads
are often poor.
1. Transfer a colony from the transformation plate to 500 L LB broth with the appro-
priate antibiotic.
2. Grow shaking at 37C overnight.
3. Transfer 180 L to a PCR tube.
4. Spin the tubes at a minimum of 3000 x g for 5 min.
5. Pour off the supernatant and tap the tube dry on a paper towel.
6. Repeat the spin.
7. Add 100 L of water and vortex.
8. Heat at 90100C for 10 min.
9. Spin at 15,000 x g for 5 min.
10. Remove the top 50 L of the supernatant and use for sequencing (in general 25 L
of this product is sufficient for sequencing when using a high copy number plasmid).
34
2.3.2 Protocol for Oligonucleotide-directed Internal Mutagenesis
For more information on this method and primer design considerations, see section 2.1.3.
1. PCR
Set up a 50 L mutagenesis reaction using a high fidelity polymerase such as KOD (No-
vagen) or Phusion (Thermo Fisher, New England Biolabs). A typical reaction setup and
cycling times are described below.
PCR Setup
35
Mutagenesis Application Guide
PCR Cycling Parameters for KOD DNA Polymerase PCR Cycling Parameters for Phusion DNA Polymerase
3. DpnI Digestion
After the cycling is finished, add 1 L of DpnI restriction endonuclease (New England
Biolabs) to the reaction mixture to remove the original plasmid DNA. Incubate at 37C
for 30 min.
4. Transformation
Transform 2 L of the final product into competent E. coli following the manufacturers
instructions. For DNA fragments smaller than 12 Kb, chemically competent cells such as
DH5 can be purchased or prepared in the lab. Larger plasmids require electroporation
for efficient uptake by bacteria.
Typical transformations use 25100 L of competent cells under the following conditions:
1. Place the competent cells on wet ice until completely thawed (25 min).
2. Add 25 L of the Dpn digestion product, not exceeding 10% of the competent cell
volume.
3. Incubate on wet ice for 30 min, stirring gently every 10 min. Do not pipette or vortex
the cells.
4. Place the cells in a 42C water bath for 3045 sec.
36
5. Immediately return the cells to wet ice for 2 min.
6. Add 10 volumes of SOC (e.g., for 25 L cells, add 250 L SOC) and place in a shaking
incubator at 37C for 1 hr. See below for an SOC media recipe.
7. Plate 100200 L of the reaction on 10 cm LB agar plates with the appropriate selec-
tion agent (often 100 g/mL ampicillin or 50 g/mL kanamycin).
8. Place in an incubator at 37C overnight.
5. Screening
Methods of screening for desired mutations vary depending on the size and complexity
of the mutated area. In general, Sanger sequencing using Applied Biosystems BigDye
Terminators and capillary sequencing is the preferred method. A protocol for a rapid
purification of DNA to screen colonies is listed below. Alternatively, a plasmid purifica-
tion kit such as Qiagens Plasmid Mini Kit can be used. Screen 520 colonies to ensure
identification of a correct clone. Note that primers used for sequencing should be at
least 40 bases away from the mutation site as the first few bases of sequencing reads are
often poor.
1. Transfer a colony from the transformation plate to 500 L LB broth with the appro-
priate antibiotic.
2. Grow shaking at 37C overnight.
3. Transfer 180 L to a PCR tube.
4. Spin the tubes at a minimum of 3000 x g for 5 min.
5. Pour off the supernatant and tap the tube dry on a paper towel.
6. Repeat the spin.
7. Add 100 L of water and vortex.
8. Heat at 90100C for 10 min.
9. Spin at 15,000 x g for 5 min.
10. Remove the top 50 L of the supernatant and use for sequencing (in general 25 L
of this product is sufficient for sequencing when using a high copy number plasmid).
37
Mutagenesis Application Guide
3. Troubleshooting
Mutagenesis is a multi-step process that varies greatly depending on the particular
method you choose, the goal of the project, and the information you have about the
target sequence. As a result, troubleshooting may be necessary in order to maximize
the desired results. Here we list some of the more common issues that arise with site-
directed mutagenesis. See the table below for the commonly observed problems and
the potential solutions to consider, and then refer to the indicated section to learn more
about the issues and how to correct them. You may need to look further into some of
these issues than is covered in the scope of this guide. We recommend two additional
resources for more information: Molecular Cloning: A Laboratory Manual [24] and Cur-
rent Protocols in Molecular Biology [25].
Observed Issue
satellite colonies
rearrangements
No PCR Product
38
3.1 Primer Design
3.1.1 Good Primer Design
Poorly designed primers can result in a lack of full-length amplification, amplification
of multiple products, or no amplification at all. See Section 2.1 for more information on
designing primers. Make sure the primers match the target sequence and include the
following primer characteristics:
39
Mutagenesis Application Guide
40
3.3 Reaction Setup
3.3.1 Experimental Setup
A failed experiment could be caused by a component inadvertently being left out of the
reaction or due to an expired reagent. To minimize pipetting variation and the chance
of leaving a reagent out of a reaction, we recommend creating a master mix of reagents
common to all reactions in the experiment. Typically the master mix will contain every-
thing except the primers and template.
We recommend that you always include a positive control, preferably with an amplicon of
similar size to the experimental PCR amplicon, and a non-template negative control. Re-
peat a failed experiment at least once to make sure that all components are included. If the
positive control does not work, repeat this control while replacing individual components
to identify the problematic reagent before trying to optimize the experimental reaction.
Prior to running any PCR, confirm that you have good quality template (see Section 3.2)
by visualizing it on an agarose gel. For more information on controls, see Section 2.2.
After running any PCR, it is critical to confirm the full length product is present before
proceeding to the next step. Run part of the reaction on an agarose gel (Figure 7). Reac-
tions that do not produce a single clean band on a gel may require either gel purification
or PCR optimization.
41
Mutagenesis Application Guide
3.3.2 Kits
Kits are optimized for use with specific buffers and enzymes. Be sure to use the
correct reagents at the suggested concentrations. Note that buffers from different
kits are not always interchangeable because the activity of each enzyme is optimal
under specific salt and pH conditions.
3.3.3 Controls
Good controls are an essential component to effective troubleshooting. At a mini-
mum each PCR should include a positive control that is known to amplify under the
same conditions as the unknown and a negative control that lacks template to test
for contamination. See Section 2.2 for details on the types of controls to use.
3.4.1 Polymerases
The use of high-fidelity polymerases is preferred over Taq polymerase or other
lower fidelity polymerases. High-fidelity polymerases decrease the number of PCR-
induced errors such as point mutations. When using the Oligonucleotide-directed
Internal Mutagenesis technique (see Section 2.1.3), it is essential to use a non-
strand-displacing polymerase such as KOD (Novagen), Phusion (Thermo Fisher,
New England Biolabs), or Pfu Turbo (Stratagene), and to avoid strand-displacing
polymerases such as Taq, Vent, and Deep Vent.
42
3.5 PCR Reaction Parameters
3.5.1 Cycle Number
Too few PCR cycles may not amplify enough product to be visible on a gel while too
many cycles may allow non-specific amplification and increase the chances of poly-
merase errors that could result in point mutations or small deletions. For site-directed
mutagenesis, it is best to use the minimum number of cycles needed to produce a
detectable band when 1/10th of the reaction is analyzed by agarose gel electrophore-
sis. Increase or reduce the number of cycles by 35 cycles at a time to find the optimal
cycle number.
43
Mutagenesis Application Guide
Create a PCR profile with an annealing temperature 510C above the predicted an-
nealing temperature of the primers and decrease it by 1C per cycle for the first 510
cycles. Follow with 2030 cycles at the lowest annealing temperature.
3.6 Ligation
3.6.1 Quantification of Product
Using the correct concentration of PCR product is critical for ligation reactions. If the
ratio of insert to vector is suboptimal, most of the resultant colonies will be either vector
with no insert or other deleted forms of the vector. A concentration that is too low will
not produce sufficient product to provide enough colonies for screening. A concentra-
tion that is too high (above 10 g/mL) may favor intermolecular ligation of multiple
plasmids, resulting in large products when run on a gel and poor transformation yields.
If you observe this, decrease the concentration of the PCR product added to the ligation
reaction by 210X.
44
3.6.2 Gel Confirmation of a Ligation Reaction
The success of a ligation reaction can be assessed by running the product on an aga-
rose gel (Figure 8). In general, supercoiled products migrate through the gel faster
than linear products. Run half of the ligation reaction in a lane next to the linear PCR
fragment to determine if a mobility shift has occurred. Note that ligation reactions
rarely reach completion so expect the presence of some linear DNA. The high salt
content of ligase buffers can also affect the mobility of the product (Figure 9). Purify
the product with a NAP5 or similar column if the product shows bands with smears.
1 2 3
1 2 3
45
Mutagenesis Application Guide
High levels of salts (Figure 10)Prior to ligation, desalt the DNA with a clean up
kit that has a size exclusion column.
Degraded ATP in the reaction bufferAliquot small volumes of the ligase buffer
to avoid repeated freeze-thaw cycles.
The use of deoxyribose ATP instead of ribose ATPNucleotides for PCR are not
an energy source for ligase.
An incubation time that is too shortBlunt-ended ligations often require 2
hours at room temperature or overnight at 16C for maximum efficiency.
A high degree of secondary structure near the ligation pointthis can cause
deletions and/or rearrangements in the vicinity of the ligation point as well as
poor ligation efficiency.
Poor storage conditions (such as storing in a frost-free freezer) or multiple
freeze-thaw cycles.
1 2 3
46
3.7 DpnI Digestion
3.7.1 Controls
The DpnI digestion can be confirmed by transforming equal amounts of digested and
undigested plasmid. An efficient digestion reaction should decrease the number of col-
onies by 12 orders of magnitude (Figure 11). This control should have the same salts
and buffers as the experimental reaction. As with many enzymes, DpnI will become inac-
tive when stored at warm temperatures. Store the enzyme in a -20C freezer.
Figure 11. Effect of DpnI Treatment. The extension product from a site-directed mutagenesis reaction was ei-
ther transformed directly or treated with DpnI and then transformed. All transformations were into chemically
competent bacteria. Colonies shown in plate 1 were derived from reactions treated with DpnI while colonies in
plate 2 were from reactions not treated with DpnI. Note the significantly fewer number of colonies in the DpnI-
treated sample. The extra colonies in plate 2 contain the original plasmid DNA without the mutation of interest.
47
Mutagenesis Application Guide
3.8 Transformation
3.8.1 Handling Competent Cells
Frozen competent cells are very fragile and are sensitive to temperature changes. Once
thawed, these cells should not be refrozen as a large loss in competency is likely to occur.
After thawing, keep cells on wet ice and use them immediately. Rough handling, includ-
ing rapid pipetting and vortexing, will also result in a loss in competency.
An antibiotic concentration that is too high can cause death of antibiotic-resistant cells
and result in no growth on plates.
48
are plated. Antibiotics degrade over time and are sensitive to heat. Low concentrations
of some antibiotics, such as ampicillin, can lead to the growth of satellite colonies. This
is because low concentrations of ampicillin are bacteriostatic rather than bactericidal.
Beta lactamase, the protein that confers ampicillin resistance, is secreted from cells that
express it. As a result, bacterial colonies that lack ampicillin resistance, and are not killed,
can grow after the formation of resistant colonies. These smaller satellite colonies lack
plasmids and often outnumber the plasmid-containing colonies.
3.8.5 Contamination
Observation of differently colored cells, filamentous growth, or inhibition of E. coli growth
indicates contamination. Replace the media and practice sanitary techniques to avoid this.
49
Mutagenesis Application Guide
4. References
1. Shortle D, DiMaio D, et al. (1981) Directed mutagenesis. Annu Rev Genet, 15: 265294.
2. Zoller MJ. (1991) New molecular biology methods for protein engineering. Curr
Opin Biotechnol, 2(4): 526531.
3. Reikofski J, and Tao BY. (1992) Polymerase chain reaction (PCR) techniques for site-
directed mutagenesis. Biotechnol Adv, 10(4): 535547.
5. Mullis KB and Faloona FA. (1987) Specific synthesis of DNA in vitro via a polymerase-
catalyzed chain reaction. Methods Enzymol, 155: 335350.
6. Ho SN, Hunt HD, et al. (1989) Site-directed mutagenesis by overlap extension using
the polymerase chain reaction. Gene, 77(1): 5159.
7. Lee J, Shin MK, et al. (2010) Insertion and deletion mutagenesis by overlap extension
PCR. In: Braman J (editor) Methods Mol Biol New York: Humana Press. 634: 137146.
10. Erster O and Liscovitch M. (2010) A modified inverse PCR procedure for insertion,
deletion, or replacement of a DNA fragment in a target sequence and its application
in the ligand interaction scan method for generation of ligand-regulated proteins.
In: Braman J (editor) Methods Mol Biol New York: Humana Press. 634: 157174.
11. Smith M. (1985) In vitro mutagenesis. Annu Rev Genet, 19: 423462.
12. Wells JA, Vasser M, et al. (1985) Cassette mutagenesis: an efficient method for gen-
eration of multiple mutations at defined sites. Gene, 34(23): 315323.
13. Georgescu R, Bandara G, et al. (2003) Saturation mutagenesis. Methods Mol Biol, 231:
7583.
50
14. You L and Arnold FH. (1996) Directed evolution of subtilisin E in Bacillus subtilis to
enhance total activity in aqueous dimethylformamide. Protein Eng, 9(1): 7783.
15. Kuchner O and Arnold FH. (1997) Directed evolution of enzyme catalysts. Trends
Biotechnol, 15(12): 523530.
16. Eckert KA and Kunkel TA. (1990) High fidelity DNA synthesis by the Thermus
aquaticus DNA polymerase. Nucleic Acids Res, 18(13): 37393744.
17. Eckert KA and Kunkel TA. (1991) DNA polymerase fidelity and the polymerase
chain reaction. PCR Methods Appl, 1(1): 1724.
18. Cadwell RC and Joyce GF. (1992) Randomization of genes by PCR mutagenesis.
PCR Methods Appl, 2(1): 2833.
19. Moore GL and Maranas CD. (2000) Modeling DNA mutation and recombination
for directed evolution experiments. J Theor Biol, 205(3): 483503.
20. McCullum EO, Williams BA, et al. (2010) Random mutagenesis by error-prone PCR.
In: Braman J (editor) Methods Mol Biol New York: Humana Press. 634: 103109.
21. Mondon P, Grand D, et al. (2010) Mutagen: a random mutagenesis method pro-
viding a complementary diversity generated by human error-prone DNA poly-
merases. In: Braman J (editor) Methods Mol Biol New York: Humana Press. 634:
373386.
22. Chiang LW, Kovari I, et al. (1993) Mutagenic oligonucleotide-directed PCR ampli-
fication (Mod-PCR): an efficient method for generating random base substitu-
tion mutations in a DNA sequence element. PCR Methods Appl, 2(3): 210217.
23. Lai YP, Huang J, et al. (2004) A new approach to random mutagenesis in vitro.
Biotechnol Bioeng, 86(6): 622627.
24. Sambrook J and Russell DW, editors. (2001) Molecular Cloning: A Laboratory
Manual. 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
25. Ausubel FM, Brent R, et al., editors. (2011) Current Protocols in Molecular Biol-
ogy John Wiley & Sons.
26. Saida F, Uzan M, et al. (2006) Expression of Highly Toxic Genes in E. coli: Special
Stragegies and Genetic Tools. Current Protein and Peptide Science, 7: 4756.
51
Mutagenesis Application Guide
Index G
Gene construction 16, 20
A I
Additions 7, 9, 10, 11, 23, 27, 31 IDT
Terminal additions 7, 9, 23, 27, 31 Oligonucleotide quality 22
Agarose gel 40, 41, 44, 45, 46 Reagents for mutagenesis 20
Annealing temperature 25, 26, 27, 39, 43, 44 Custom Mixed Bases 21
Antibiotics 48, 49 Genes 16, 20
Ampicillin 48, 49 Machine Mixed Bases 21
Kanamycin 48 Phosphate Modifications 20
Primers 20
C Trimers 21
Cassette mutagenesis. SeeMutagenesis Ultramer Oligonucleotides 20, 25
Cassettes 11, 16, 17 Universal bases 21
Chemical mutagenesis. SeeMutagenesis In vitro saturation. SeeRandom mutagenesis
Cloning 5, 6, 12, 16, 20, 22, 23. See alsoTransfor- Inverse PCR 6, 7, 12, 13, 14, 15
mation
Competent cells 29, 30, 33, 36, 48, 49 K
Contamination 42, 49 Kits 34, 37, 42
Controls 29, 30, 41, 42, 47 KOD. SeePolymerase
DpnI digestion controls 30, 47
Ligation controls 30 L
PCR controls 29, 41, 42 Libraries 16, 18, 23
Transformation controls 30 Ligation 13, 14, 15, 30, 32, 44, 45, 46
Ligase 30, 32, 46
D
Dam methylase. SeeMethylated DNA M
ddRNAi 20 Melting temperature (Tm) 25, 26, 27, 28, 39, 42
Degenerate primers 21. SeePCR with degenerate Methylated DNA 12, 47
primers Mispriming 7
Deletions 6, 7, 9, 10, 11, 43, 46, 49 Mixed bases 19, 21
Denaturation temperature 44 MutaGen 18
DeoxyInosine 21 Mutagenesis
DH5. SeeCompetent cells Cassette mutagenesis 16, 17
Directed protein evolution 16 Chemical mutagenesis 19
DpnI. SeeRestriction enzyme Random mutagenesis 5, 16, 19
Saturation mutagenesis 19
E Semi-random mutagenesis 19
E. coli. SeeCompetent cells Site-directed mutagenesis 5, 6, 7, 10, 12, 16, 22,
Electroporation 33, 36, 48 23, 24, 28, 39, 41, 43, 47
Enzymatic mutagenesis. SeeError-prone PCR Mutator strains 18
Error-prone PCR 6, 18
Ethyl methane sulfonate (EMS) 19 N
Nucleotide analogs 18
52
O Restriction enzyme 12, 16, 17, 26, 46
OligoAnalyzer 3.1 25, 26, 27, 39 DpnI 12, 24, 30, 32, 33, 36, 38, 47
Oligonucleotide-directed Internal Mutagenesis Restriction site 6, 12, 16, 23, 26
23, 28, 31, 35, 42 S
P Saturation mutagenesis. SeeMutagenesis
PCR. See alsoError-prone PCR; See alsoInverse SciTools 25, 39. See alsoOligoAnalyzer 3.1; See
PCR; See alsoTouchdown PCR; See alsoUNAFold
alsoTerminal additions by PCR; See Screening for mutants 16, 29, 34, 37, 44
alsoTerminal changes by PCR Semi-random mutagenesis. SeeMutagenesis
Cycling conditions. SeePCR Reaction param- Sequencing 34, 37
eters Site-directed mutagenesis. SeeMutagenesis
PCR with Degenerate Primers 19 SNP 6
Primer design 10, 25, 27, 28, 39 SOC media 33, 37
Reaction parameters 25, 29, 43 Substitutions 7, 8, 19
Phusion. SeePolymerase T
Plasmid 12, 13, 14, 15, 16, 17, 28, 29, 30, 32, 34,
36, 37, 40, 41, 43, 44, 45, 46, 47, 49 T4 DNA Ligase 32. See alsoLigation
Plasmid purification 34, 37, 44 Taq DNA polymerase. SeePolymerase
Polymerase 12, 18, 19, 20, 21, 24, 31, 32, 35, 36, Terminal additions by PCR 23, 27, 31
Terminal changes by PCR 23, 26, 31
40, 42, 43, 44
Touchdown PCR 44
High fidelity 12, 31, 32, 35, 36
KOD DNA Polymerase (Novagen) 24, 26, 31, Toxic sequences 49
32, 35, 36, 42 Transformation 30, 33, 36, 48
Pfu Turbo (Stratagene) 35, 42 Trimers 21
Phusion (Thermo Fisher, New England Bio- U
labs) 31, 32, 35, 36, 42
Ultramer Oligonucleotides 5, 6, 7, 9, 10, 16, 20,
Taq DNA polymerase 18, 32, 35, 42
23, 24, 25, 28
Primer extension 6, 7, 10, 11, 12
UNAFold 39
Protocols 31
Universal bases 21
Protocol for oligonucleotide-directed internal
UV irradiation 18
mutagenesis 35
Protocol for terminal changes or additions 31 V
Q Vector 5, 16, 44, 46. See alsoPlasmid
Quality
Oligonucleotide 5, 7, 16, 20, 22, 25, 40, 41
Template 40
Quick Ligation Kit (New England Biolabs) 32
QuikChange Site-Directed Mutagenesis Kit
(Stratagene) 12, 28, 39
R
Random mutagenesis. SeeMutagenesis
Restriction digest 30, 32, 36, 38, 46, 47
53
Mutagenesis Application Guide
54