8 Approaches to Random Mutagenesis

Nick Oswald
Tech Tips
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Random mutagenesis is an incredibly powerful tool for altering the properties of

enzymes. Imagine, for example, you were studying a G-protein coupled receptor (GPCR)
and wanted to create a temperature-sensitive version of the receptor or one that was
activated by a different ligand than the wild-type. How could you do this?

Firstly, you would clone the gene encoding the receptor, then randomly introduce
mutations into the gene sequence to create a “library” containing thousands of versions of
the gene. Each version (or “variant”) of the gene in the library would contain different
mutations and so encode receptors with slightly altered amino acid sequences giving
them slightly different enzymatic properties than the wild-type.

Next, you could transform the library into a strain where the receptor would be expressed
and apply a high throughput screen to pick out variants in the library that have the
properties you are looking for. Using a high throughput screen for GPCR activity (see
here for examples) you could pick out the variants from the library that were temperature-
sensitive or were activated by different ligands.

Sound easy? Well, of course it’s not that easy. Creating a random mutant library that
contains enough variants to give you a good chance of obtaining the altered enzyme you
desire is a challenge in itself. There are many ways to create random mutant libraries,
each with it’s own pros and cons. Here are some of them:

1. Error-prone PCR. This approach uses a “sloppy” version of PCR, in which the
polymerase has a fairly high error rate (up to 2%), to amplify the wild-type sequence. The
PCR can be made error-prone in various ways including increasing the MgCl2 in the
reaction, adding MnCl2 or using unequal concentrations of each nucleotide. Here is a
good review of error prone PCR techiques and theory. After amplification, the library of
mutant coding sequences must be cloned into a suitable plasmid. The drawback of this
approach is that size of the library is limited by the efficiency of the cloning step.
Although point mutations are the most common types of mutation in error prone PCR,
deletions and frameshift mutations are also possible. There are a number of commercial
error-prone PCR kits available, including those from Stratagene and Clontech.

2. Rolling circle error-prone PCR is a variant of error-prone PCR in which wild-type

sequence is first cloned into a plasmid, then the whole plasmid is amplified under error-
prone conditions. This eliminates the ligation step that limits library size in conventional
error-prone PCR but of course the amplification of the whole plasmid is less efficient
than amplifying the coding sequence alone. More details can be found here.

3. Mutator strains. In this approach the wild-type sequence is cloned into a plasmid and
transformed into a mutator strain, such as Stratagene’s XL1-Red. XL1-red is an E.coli
strain whose deficiency in three of the primary DNA repair pathways (mutS, mutD and
mutT) causes it to make errors during replicate of it’s DNA, including the cloned plasmid.
As a result each copy of the plasmid replicated in this strain has the potential to be
different from the wild-type. One advantage of mutator strains is that a wide variety of
mutations can be incorporated including substitutions, deletions and frame-shifts. The
drawback with this method is that the strain becomes progressively sick as it accumulates
more and more mutations in it’s own genome so several steps of growth, plasmid
isolation, transformation and re-growth are normally required to obtain a meaningful
library.

4. Temporary mutator strains. Temporary mutator strains can be built by over-

expressing a mutator allele such as mutD5 (a dominant negative version of mutD) which
limits the cell’s ability to repair DNA lesions. By expressing mutD5 from an inducible
promoter it is possible to allow the cells to cycle between mutagenic (mutD5 expression
on) and normal (mutD5 expression off) periods of growth. The periods of normal growth
allow the cells to recover from the mutagenesis, which allows these strains to grow for
longer than conventional mutator strains.

If a plasmid with a temperature-sensitive origin of replication is used, the mutagenic

plasmid can easily be removed restore normal DNA repair, allowing the mutants to be
grown up for analysis/screening. An example of the construction and use of such a strain
can be found here. As far as I am aware there are no commercially available temporary
mutator strains.

5. Insertion mutagenesis. Finnzymes have a kit that uses a transposon-based system to

randomly insert a 15-base pair sequence throughout a sequence of interest, be it an
isolated insert or plasmid. This inserts 5 codons into the sequence, allowing any gene
with an insertion to be expressed (i.e. no frame-shifts or stop codons are cause). Since the
insertion is random, each copy of the sequence will have different insertions, thus
creating a library.
6. Ethyl methanesulfonate (EMS) is a chemical mutagen. EMS aklylates guanidine
residues, causing them to be incorrectly copied during DNA replication. Since EMS
directly chemically modifies DNA, EMS mutagenesis can be carried out either in vivo
(i.e. whole-cell mutagenesis) or in vitro. An example of in vitro mutagenesis with EMS in
which a PCR-amplified gene was subjected to reaction with EMS before being ligated
into a plasmid and transformed can be found here.

7. Nitrous acid is another chemical mutagen. It acts by de-aminating adenine and

cytosine residues (although other mechanisms are discussed here) causing transversion
point mutations (A/T to G/C and vice versa). An example of a study using
nitrosoguanidine mutagenesis can be found here.

Note: I have only mentioned two chemical mutagens but there are many others. Hirokazu
Inoue has written an excellent article describing some of them and their use in
mutagenesis, see here (pdf).

Another note: Chemical mutagens are, of course… mutagens and therefore should be
handled with great care. Be especially careful with EMS as it is volatile at room
temperature. Read the MSDS and do a proper risk assessment before carrying out these
experiments.

8. DNA Shuffling is a very powerful method in which members of a library (i.e. copies
of same gene each with different types of mutation) are randomly shuffled. This is done
by randomly digesting the library with DNAseI then randomly re-joining the fragments
using self-priming PCR. Shuffling can be applied to libraries produced by any of the
above method and allows the effects of different combinations of mutations to be tested.
For more information see here and here (see page 13).

In Vitro Mutagenesis:
Natural mutation, in general, is spontaneous. Any
heritable change is considered as mutation. It can be
spontaneous or induced. Mutation cannot be predicted
in nature. Mutation at chromosomal level can be
numerical (ploidy) or it can be structural (aberrations).
At molecular level mutation can be deletion of a
sequence of nucleotide or nucleotides, or addition of
nucleotide or nucleotides, translocation of DNA
segments with in the chromosome or between
chromosomes or it can be due to inversion of DNA
segments.
Point mutations are generally restricted to changes in a
single nucleotide i.e. substitution of a nucleotide or
addition of nucleotide or loss of a single nucleotide.

Mutation can lead to gain of a function called Gain

mutation, or loss of a function called null mutation.
Mutation can conditional, where the effect of mutation
is expressed or manifest only under certain condition.
Many mutations are spontaneously reversed called
reverse mutations, but some mutations are suppressed
called suppressor mutations.

The rate of mutations varies form one organism to

another, which depends upon the phylogenetic and
hierarchical position of the organism. In that sense
viruses show greater propensity for changes than
Bacterial systems. And bacterial systems are prone to
mutations than higher organisms. Haploids suffer more
than diploids due to mutation of one or the other kind.

All natural mutations are unpredictable and spontaneous

and it is the primary force of nature that caused
variations and those variations survived the test of the
time are fixed and furthered, thus species originated in
nature. Today molecular technology that is available at
hand can be used create desirable mutation under in
vitro condition. It is not just creating random
mutations; it is now possible to create mutations to
create new codons, new messages and new
characters. This is possible to target a single
nucleotide or a group of nucleotides. In this process
one can change a single codon or add one or more
codons and delete one or more codons. Such changes
in coding sequence of a particular gene can generate a
protein with new conformation or new function or both;
in essential it amounts to protein engineering.

Protein engineering is an emerging technology of great

importance in medicine and industry. By this
technology one can enable the protein to be stable at
 higher temperature and still active in non-aqueous
conditions. One can make the protein to change its
affinity to the substrate and increase the activity by
several folds at low Km. It is also possible to change its
specificity of the substrate and function. This is going
to be one of the futuristic aspects of biotechnology.

Application of Site Directed

Mutagenesis:
• Proteins stable and functional at high temperatures.

• Proteins stable and functional at changed pH.

• Proteins active in non-aqueous solvents.

• Proteins don’t require cofactors for their function.

• Proteins resistance to proteases.

• Proteins with changed allosteric features.

• Proteins with changed active site and increased

specific activity.

• Proteins with changed Km and Vmax with increased

catalytic efficiency.

A list of few engineered proteins:

• Alpha Amylase- used in bear making.

• Subtisilin-biodetergent.

• Amino cyclase- Preparation of L-amino acids.

• Bromelain- Meat tenderizer and juice clarifier.

• Catalase- anti oxidant in food preparation.

 • Ficin- meat tenderizer.

• Cellulase- alcohol and glucose production.

• Gluco amylase- beer making and other EtOH

products.

• Glucose oxidase- anti oxidant in food processing.

• Invertase- sucrose inversion.

• Lipase- cheese making and flavoring.

• Papain- meat tenderizer and juice clarifier.

• Pectinase- juice clarifier.

• Protease- detergent preparation.

• Rennet- cheese making.

How proteins are engineered:

• Thermo stable T4 Lysosome: - Changing
few amino acids to cysteine that produce more disulfide
bonds make the proteins resistant to higher
temperatures. Addition of cysteine does not change
the 3-D structure and the function of the protein, yet it
is stable at high Tm.

• Thermo stable Triose phosphate isomerase:

Increase in temperature leads to deamidation of
Aspargine and glutamine. This deforms the protein and
so it looses its activity at higher Tm. By in vitro site
directed mutagenesis amino acid Aspargine at 14 and
78 are changed to Threonine or Isoleucine. These
changes have made the proteins to be thermo stable.
 • Cloned b-interferon: Interferons produced in bacteria
were not active because of dimerization and
multimerization. The proteins were found to be
inactive. By in vitro mutagenesis one or two cysteine
were changed to serine. This prevents the individual
subunits from dimerization or multimerization, yet they
are found to be active.

• Active site modification: Tyrosyl tRNA synthase is

the enzyme responsible for adding tyrosine to the
tRNA. Changing the Threonine at 51-st position to
Proline has made the enzyme to have increased
specific activity and increased specificity.

• Changed Staphylococcus nuclease (S1 nuclease);

The S1 nuclease recognizes both ss RNA and ssDNA
and dsDNA and acts at A.U or A.T rich regions and cuts
to generate 3’phosphate and 5’OH groups. When
Lysine at 116 positions was changed to cysteine, the
protein showed remarkable site-specific activity.

• Subtisilin: It is a bacterial protein used in detergents.

This protein is responsible for removing proteinaceous
dirt. But the protein is susceptible for bleach and it is
rendered inactive. By changing and addition of one or
two cysteine residues, the number of S-S bonds
increase. This had made the proteins to be stable not
only in the detergent but also stable for bleach. Such
engineered protein is used in detergent powder called
biodetergent.

In Vitro Random Mutation:

First method:
Using chemicals creates mutation, but the site of mutation is
random. Whether or not in vitro mutation has any desirable
feature is perhaps a chance.
In order to induce mutations, obtain ssDNA from M13/18
plasmids with an insert gene of interest.

The ssDNA is subjected very low level of sodium bisulfate

under controlled conditions such that the number of site
mutated will be very very minimal. Sodium bisulfate
deaminates cytosine residues to uracil. When the DNA
containing uracil replicates it produces DNA with Adenine in
place of uracil. Thus whatever limited numbers of cytosine
residues converted into adenines change the message, so
also the protein product.

Such deaminated and oxygenated ssDNA is copied into

dsDNA by using an universal primer. Such replicative form
DNA is used for bacterial transformation. Then the colonies
are screened for any mutation. This way one can generate a
large number of point mutation.

Second method:

Some of the E.coli strains such as Ung ( – )and Dut(+) are very
useful obtaining in vitro mutation, again it is random. Ung –
strains are incapable of removing uracil nucleotides from the
DNA by deglycosylation reaction. Dut – strains are lacking
UTPase, thus the concentration of UTP inc5reases and UTPs
are incorporated into the DNA. But Ung+ and Dut + strains
have functional enzymes.

If a M13/18 plasmid containing a specific gene is used to

transform double mutant strain, UTPs are incorporated into
the replicating DNA and incorporated UTPs are not removed
by N’deglycosylases, because of defective enzymes.

Obtain positive ssDNA from M13/18 transformed strains and

using universal primers generate ds DNA.

Digest the plasmid in order to release the insert and clone

the same into another plasmid and transform Ung + and Dut
– bacterial strains. As the plasmid DNA replicates all the Us
are removed by deglycosylases and the replicated plasmid is
obtained.

Obtain the plasmids and release the insert and recline the
gene and analyze for the random mutants.

Site directed Mutagenesis:

If the sequence of the desired DNA is known, from that one
can find out certain restriction enzyme sites. For example
within the gene of interest, assume there is one E.coR I site;
the sequence of it is GAATTC.

If the site is cut with EcoR I enzyme it generates 5’

overhangs in both strands of the DNA.

----5’G AATTC---3’

----3’CTTAA G---5’

If such DNA with sticky ends is treated with S1 nuclease it

removes the overhangs to blunt ends.

-----5’G C---3’

-----3’C G----5’

When such ends are ligated one gets the DNA with 5’GC3’
sequences and four base pairs are lost. This leads not only
to deletions but also changes the reading frame.

Similarly if the cut ends are treated with T4 DNA polymerase

with dNTPs, the enzyme fills the gaps of each ends by
extending the cut ends by 5’3’ direction to produce blunt
ends. If such blunt ends are ligated they generate
5’GAATTAATTC3’. In this four base pars are added. Again
this causes the addition and the reading frame is changed.
Mostly this kind of changes doesn’t produce any functional
protein, if by chance the changed reading frame works you
are in luck.
Method II:

In this case the change is directed to one or more specific

nucleotides thus one can change only one of the codons. For
example one wants to change Phenyl alanine (UUU) to
Cysteine (UGU), design primers in such a way UUU is
changed to UGU, where with exception of one nucleotide
others are complementary to the nucleotides on either side
of the said nucleotide.

5’--UUU

3’—AAA

Primer to the bottom strand- wit5h changed

nucleotide-----5’UGU

Primer to the top strand with changed nucleotide

----- -<-ACA 5’

Melt the DNA to ssDNA and anneal the primer to the strands
not to a state of stringency. This will provide an opportunity
for the base pairs, which are not complementary, remain
unpaired. Amplify the DNA using PCR protocols. Then melt
the DNA to single strands and anneal them at high Tm for
stringency.

Then use the plasmids and transform them and look for the
change in the codon.

Another way is to have marker in the DNA which one wants

to use for site directed mutation. Take a plasmid with the
desired gene and a marker out side the gene such as a
restriction site which can be cut with only Dpn-I. Use the
primers and amplify the DNA. Then melt the DNA and
Anneal. The parental strands anneal and new strands
anneals by themselves. After annealing the DNA, cut the
dsDNA with Dpn-I. The new strands have no Dpn-I site for
the enzyme requires methylated sites, otherwise it does not
recognize the site. Thus the parental strands are cut, but
not the new strands. When such DNA is used for bacterial
transformation the cut DNA cannot transform and the uncut
DNA transforms bacteria, thus one obtains the DNA with
changed sequence to generate a new codon

Primer extension

BIOC/MCB 568 -- Fall 2010

John W. Little--University of Arizona
BIOC/MCB568 Home Page

Methods

Primer extension is used to map the 5' ends of DNA or RNA fragments. It is done by
annealing a specific oligonucleotide primer to a position downstream of that 5' end. The
primer is labeled, usually at its 5' end, with 32P. This is extended with reverse
transcriptase, which can copy either an RNA or a DNA template, making a fragment that
ends at the 5' end of the template molecule. DNA polymerase can also be used with DNA
templates.

Uses:

1. Mapping the 5' end of transcripts. This allows one to determine the startpoint of
transcription (assuming the mRNA isn't further processed), which helps localize
promoters or TATA boxes.

2. Quantifying the amount of transcript in an in vitro transcription system. An example is

the Yudkovsky et al. paper.
3. Determine the locations of breaks or modified bases in a mixed population of RNA or
DNA samples. This is useful in applications like footprinting. Two different methods are
used. In one, the modified nucleotide cannot be recognized by the polymerase or reverse
transcriptase; in such cases, the chain ends at the site of modification (as with KMnO4 --
for instance in the Kainz and Roberts paper). In the other, the modification is converted
in a later step of the analysis to a strand break by chemical treatment. For instance, the
sites of modifications by dimethyl sulfate (DMS) can be identified by treating DNA with
DMS, exposing the sample to conditions that break the backbone at the site of
modification, followed by primer extension.