Ma’am Afshan Bukhari

• A heritable change in the base sequence of DNA.

• A change that occurs in our DNA sequence, either due to
mistakes when the DNA is copied or as the result of
environmental factors such as UV light etc.

• Point mutation
• Additions
• Deletions
• Substitutions
• Frame-shift mutation
• Other mutations…
• Point Mutation:
a change in one base in the DNA sequence.

• Substitution:
when one or more bases in the sequence is replaced by the
same number of bases (for example, a cytosine substituted for
an adenine)
• Inversion :
when a segment of a chromosome is reversed end to end.

• Insertion:
when a base is added to the sequence.
• Deletion:
when a base is deleted from the sequence.

• Copy number variation: (CNV)

it is a type of mutation where large chunks of DNA are
inserted, repeated or lost. These regions of DNA can be
between 10,000 and 5,000,000 bases long.
• Silent Mutation: base change, no amino acid change

• Neutral Mutation: Base change resulting in amino acid

change that does not affect protein function
• EX. Aspartic acid (D) Glutamic acid (E)

• Missense mutation: altered codon, new amino acid with

different chemical properties. Function is affected.

• Nonsense mutation: base pair substitution results in a

stop codon (and shorter polypeptide)

• Frame-shift mutations: additions or deletions.

Peptide may be longer or shorter.
• Incorrect base pairing due to tautomeric shifts
• Removal of nitrogenous bases
• Alteration of nitrogenous bases
• Addition or deletion of nucleotides
• Single strand breaks
• Double strand breaks
• Crosslinking—covalent linkage between bases
• DNA damage is an abnormal alteration in the structure
of DNA that cannot, itself, be replicated when DNA replicates.
• In contrast, a mutation is a change in the nucleic acid
sequence that can be replicated; hence, a mutation can
be inherited from one generation to the next.
• When DNA containing damage is replicated, an incorrect base
may be inserted in the new complementary strand as it is being
synthesized.
• The incorrect insertion in the new strand will occur opposite to
the damaged site in the template strand, and this incorrect
insertion can become a mutation (i.e. a changed base pair)
• A process by which the genetic information of an organism is
changed, resulting in a mutation.
• It may occur spontaneously in nature, can lead to cancer and
various heritable diseases, but it is also a driving force of
evolution.
• It can be introduced in specific ways as a result of exposure to
mutagens, experimentally using laboratory procedures and then
observing the phenotype of the organism the function of genes
and even individual nucleotides can be determined.
• It can be used as a powerful genetic tool. E.g. in Protein
Engineering, Error-free Cloning, Gene Editing, Mutational
Analysis
• It is a well-established assembly reaction that can be leveraged
to join multiple, mutagenized DNA fragments with overlapping
ends.
• The inherent flexibility of this approach lends itself to small and
large constructs and encompasses both single and multiple insert
assemblies.
1. Generate primers for insertion, substitution, or deletion
mutagenesis
2. Combine DNA fragments and vector for uniform assembly
using complementary overlaps between fragments.
3. Perform PCR amplification to introduce mutation(s) and
overlapping ends using GA SDM PCR Mix (2X)
4. Fragments annealing
5. GA SDM Assembly Mix B (2X) mediates extension and repair
• Accurate, seamless, and efficient mutagenesis and cloning

• Multiple mutations (up to 5) incorporated in a single round

• Accommodates large insertions (up to 40 bp)

• Allows for deletions of any size

• Optimal for 1–5 fragments

• Appropriate for linear or circular templates

• Expected Efficiency: 95% (single site), 55% (five site)

• Another use of cloned DNA is in vitro mutagenesis in which a
mutation is produced in a segment of cloned DNA. The DNA is
then inserted into a cell or organism, and the effects of the
mutation are studied.
• Mutations are useful to geneticists in enabling them to investigate
the components of any biological process. However, traditional
mutational analysis relied on the occurrence of random
spontaneous mutations—a hit-or-miss method in which it was
impossible to predict the precise type or position of the mutations
obtained.
• In vitro mutagenesis, however, allows specific mutations to be
tailored for type and for position within the gene. A cloned gene
is treated in the test tube (in vitro) to obtain the specific mutation
desired, and then this fragment is reintroduced into the living cell,
where it replaces the resident gene.
• Directed Mutagenesis/
Oligonucleotide-directed

• Site-directed/Site-specific
Mutagenesis

• Mismatched Mutagenesis
• Directed mutagenesis, also known as directed mutation, was a hypothesis
proposing that organisms can respond to environmental stresses by
orthogenetically directing mutations to certain genes or areas of the genome.
• A specific point in a sequenced gene is pinpointed for mutation.

• An oligonucleotide, a short stretch of DNA of the desired sequence, is made

chemically. For example, the oligonucleotide might have adenine in one specific
location instead of guanine. This oligonucleotide is hybridized to the
complementary strand of the cloned gene; it will hybridize despite the
one base pair mismatch.

• Various enzymes are added to allow the oligonucleotide to prime the synthesis
of a complete strand within the vector. When the vector is introduced into a
bacterial cell and replicates, the mutated strand will act as a template for a
complementary strand that will also be mutant, and thus a fully mutant
molecule is obtained. This fully mutant cloned molecule is then reintroduced into
the donor organism, and the mutant DNA replaces the resident gene.
• the resident
functional gene is
replaced by a
completely
nonfunctional copy.
The advantage of
this technique over
random mutagenesis
is that specific genes
can be knocked out
at will, leaving all
other genes
untouched by the
mutagenic
procedure.
• It is used to make intentional changes at the specific site in a cloned DNA
(sequence of a gene and any gene products), altered in a precise, pre-
determined way.
• This known sequence is used to chemically synthesize specific nucleotide
substitutions, deletions, and so on for investigating the structure and biological
activity of DNA, RNA, and protein molecules, e.g. protein engineering.
• Through single-strand hybridization, these oligonucleotides can be directed to any
chosen site in the gene.
• In one approach, the gene of interest is inserted into a single-
stranded phage vector, such as the phage M13.
• A synthetic oligonucleotide containing the desired mutation is designed.
• This oligonucleotide is allowed to hybridize to the mutant site by complementary
base pairing.
• The oligonucleotide serves as a primer for the in vitro synthesis of the
complementary strand of the M13 vector
• The basic procedure requires the synthesis of a short DNA
primer.
This synthetic primer contains the desired mutation and is
complementary to the template DNA around the mutation site so
it can hybridize with the DNA in the gene of interest.
The mutation may be a single base change (a point mutation),
multiple base changes, deletion, or insertion.
• The single-strand primer is then extended using a DNA
polymerase, which copies the rest of the gene.
The gene thus copied contains the mutated site, and is then
introduced into a host cell as a vector and cloned.
Finally, mutants are selected by DNA sequencing to check that
they contain the desired mutation.
• 5’ add-on mutagenesis
- a new sequence or chemical group is added to the 5’ end of a
PCR product

• Mismatched primer mutagenesis

• Same like site-directed mutagenesis.
• But it only focuses on single amino acid.
• Determines a particular missense mutation in a known gene of
disease.
• Evaluate the contribution of specific amino acid to the function
of protien.
• Any desired specific base change can be programmed into the
sequence of the synthetic primer. Although there will be a mis-
paired base when the synthetic oligonucleotide hybridizes with the
complementary sequence on the M13 vector, a few mismatched
bases can be tolerated when hybridization takes place at a low
temperature and a high salt concentration.
• After DNA synthesis has been mediated by DNA polymerase in
vitro, the M13 DNA is allowed to replicate in E. coli,in which case
many of the resulting phages will be the desired mutant. The
synthetic oligonucleotide can be used as a labeled probe to
distinguish wild-type from mutant phages.
• Although, at low temperature, the mismatched base will not prevent
the primer from hybridizing with both types of phage, at high
temperature, the primer will hybridize only with the mutant phage.
Oligonucleotides with deletions or insertions will direct comparable
mutations in the resident gene.
• The site-directed method can also be used on genes cloned in
double-stranded vectors if the DNA is first denatured.
• A knowledge of restriction sites is also useful in modifying a
cloned gene. For example, a small deletion can be made by
removing the fragment liberated by cutting at two restriction sites
(Figure 13-1b).
• With the use of a similar double cut, a fragment, or “cassette,” can
be inserted at a single restriction cut to create a duplication or
other modification (Figure 13-1c). Another approach is to
enzymatically erode a cut end created by a restriction enzyme, to
create deletions of various lengths (Figure 13-1d).
• The polymerase chain reaction (PCR) also can be used (Figure 13-
1e). A primer containing a mutation is used in a first round of PCR.
The product of the first round is used as a primer for a second
round of PCR, whose product is the mutant gene.
• An effective strategy for improving or altering the activity of
biomolecules for industrial, research and therapeutic applications.
• Over many generations, iterated mutation and natural selection
during biological evolution provide solutions for challenges that
organisms face in the natural world. However, the traits that result
from natural selection only occasionally overlap with features of
organisms and biomolecules that are sought by humans.
• To guide evolution to access useful phenotypes more frequently,
humans for centuries have used artificial selection, beginning with the
selective breeding of crops1 and domestication of animals2. More
recently, directed evolution in the laboratory has proved to be a
highly effective and broadly applicable framework for optimizing or
altering the activities of individual genes and gene products, which
are the fundamental units of biology.
• Researchers can use focused mutagenesis to maximize the likelihood
that a library contains improved variants, provided that amino acid
positions that are likely determinants of the desired function are
known.
• Random mutagenesis can provide a greater chance of accessing
functional library members than focusing library diversity on
incorrectly chosen residues that, when mutated, do not confer
desired activities.
• Transformation of the XL1-red strain with a plasmid bearing the
evolving gene yields mutations at a rate of 10−6 per base per
generation.
• Unfortunately, these strains not only mutate the library member but
also induce deleterious mutations in the host genome. Host
intolerance to a high degree of genomic mutation places an upper
limit on in vivo mutagenesis rates.
• Traditional genetic screens use chemical and physical agents to
randomly damage DNA. These agents include alkylating
compounds such as ethyl methanesulfonate (EMS), deaminating
compounds such as nitrous acid, base analogues such as
2-aminopurine, and ultraviolet irradiation.
• Chemical mutagenesis is sufficient to deactivate genes at
random for a genome-wide screen but is less commonly used for
directed evolution because of biases in mutational spectrum.
• Non-chemical methods to randomly mutate genes frequently
enhance the rate of errors during DNA replication.
• In error-prone PCR (epPCR), first described by Goeddel and co-
workers19, the low fidelity of DNA polymerases under certain
conditions generates point mutations during PCR amplification of
a gene of interest. Increased magnesium concentrations,
supplementation with manganese or the use of mutagenic dNTP
analogues20 can reduce the base-pairing fidelity and increase
mutation rates to 10−4~10−3 per replicated base21. Because
mutations during PCR accumulate with each cycle of
amplification, it is possible to increase the average number of
mutations per clone by increasing the number of cycles.
• One application of epPCR is to generate neutral drift libraries
that exhibits sequence diversity that does not destabilize protein
structure and is therefore largely devoid of the deleterious
mutations .
• It uses synthetic DNA oligonucleotides containing one or more
degenerate codons at positions corresponding to targeted
residues. This mutagenic oligonucleotide is incorporated into a gene
library as a mutagenic cassette using either traditional restriction
enzyme cloning or contemporary gene assembly protocols. The
simultaneous saturation mutagenesis of multiple residues can access
combinations of mutations that may exhibit epistatic interactions.
• Molecular modelling can also predict specific amino acid
substitutions that are likely to be beneficial. Algorithms such as
Rosetta calculate free energies based on steric clashes,
hydrophobic packing, hydrogen bonding and electrostatic
interactions. Mutations that are predicted to stabilize protein
folding or to improve transition state stabilization can be
introduced into the library semi-stochastically by incorporating
synthetic oligonucleotides via gene reassembly (ISOR).
• Directed evolution practitioners increasingly use sophisticated
focused mutagenesis methods to construct smaller libraries of
higher quality that sample a functionally rich portion of the fitness
landscape. These strategies require phylogenetic information or
molecular structures to focus library diversity on residues or even
specific substitutions that are thought to be necessary for the
desired activity. In the absence of this information, random
mutagenesis is an absolute necessity. Even when the requisite data
are available, deducing the determinants of protein function at the
amino acid level can be challenging. Random mutagenesis maybe
used to probe mutations that are distant from obvious substrate
contact sites or that are not present in naturally evolved
orthologues. Fortunately, random and focused mutagenesis
strategies can be combined into a single diversification step or
applied separately during successive rounds of evolution to
maximize the likelihood of success.
• When choosing a methodology, researchers should assess the
features of the protein that is being evolved to find an optimal
screening and selection technology, as well as an appropriate
accompanying genetic diversification strategy.
• Pioneering studies in the field of directed evolution sought to
improve the wild-type activity of enzymes through the
enhancement of solubility, thermostability, affinity for substrate
or catalytic turnover.
• These properties remain important in contemporary directed
evolution because increased activity and stability often
facilitate the engineering or evolution of other desirable
properties.
• New screens and selections that achieve higher throughput or
carry out more continuous rounds of evolution can broaden the
exploration of the fitness landscape, whereas novel mutagenesis
strategies increase the search efficiency.
• Through computational techniques and creative molecular
biology protocols, diversity is focused on residues and specific
mutations that influence desired activities.
• New directed evolution methods will continue to generate
proteins with useful new activities and specificities, as well as
expand the scope of protein evolution to include even larger
sets of chemical and biological functions.