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30-May-16

Uses for mutagenesis


Unit 10
Recombinant engineering of
gene function: mutagenesis

Define the role of a gene--are phenotypes


altered by mutations?
Determine functionally important regions of a
gene (in vivo or in vitro)
Improve or change the function of a gene
product
Investigate functions of non-genes, eg. DNA
regions important for regulation

Obtaining useful enzymes

Protein engineering-Why?
Enhance stability/function under new conditions
temperature, pH, organic/aqueous solvent,
Alter enzyme substrate specificity
Enhance enzymatic rate
Alter epitope binding properties

From Koeller and Wang, enzymes for chemical synthesis, Nature 409, 232 - 240 (2001)

Types of Mutagenesis
PCR Based Methods

Site-directed mutagenesis
Mismatched mutagenesis
5 add on mutagenesis
Cassette Mutagenesis

Insertional Mutagenesis
Trasposon mutagenesis

In vivo Mutagenesis
Direct Mutagenesis

Site-directed mutagenesis
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.

30-May-16

Mismatched mutagenesis
Similar to Site-Directed Mutagenesis
But only focuses on a single amino acid
Important when trying to determine a
particular missense mutation in known gene
of a disease.
Or when just trying to evaluate the
contributation of the single amino acid to the
function of the protein.

Altering the genetic code

The genetic code


61 sense codons, 3 non-sense (stop) codons
20 amino acids
Other amino acids, some in the cell (as precursors to other
amino acids), but very rarely have any been added to the
genetic code in a living system
Is it possible to add new amino acids to the code?
Yes...sort of
Wang et al. (2001) Expanding the genetic code Science 292, p.
498.

1. Mutagenesis using Using M13 phage vector

on gene or
cDNA c ci
vo

Screen for
mutant

In this way, a single-stranded DNA molecule is converted into a


double-stranded molecule, where all sequences will be wild-type
except the region containing the nucleotides. The resulting doublestranded heteroduplex DNA molecule, composed of one wild-type
strand and one mutant strand, is introduced into competent E. coli
cells, where the two strands segregate by DNA replication.
Theoretically, each colony should contain both wild-type and mutant
plasmids; in practice, however, only one sequence is often obtained
from a colony due to mismatch repair prior to replication of the two
strands. Generally, the frequency of mutant plasmids is lower than
that of the wild type. This is most likely due to incomplete DNA
replication in vitro. A number of selection or screening methods have
been developed to enrich for plasmids with the desired mutation
mismatch repair prior to replication of the two strands.

30-May-16

(Stratagene)

Primer design for


site-directed mutagenesis

5
3

*
*

3
5

5. Mega-primer
method

6. Overlapextension
method

The amplified
product from the
first round of PCR is
used as a primer in
the second round of
PCR.

WRONG?

7. Inverse PCR

Inverse PCR

Q5 Site-Directed Mutagenesis Kit

30-May-16

Inverse PCR

Q5 Site-Directed Mutagenesis Kit

8. The 5 add on Mutagenesis


Involves adding on a new sequence or
chemical group to the 5end of a PCR product
This involves a particular way of designing the
primers:
3 end of the primer matches the sequence of PCR
product.
5 end contains the novel sequence
Suitable restriction site
Addition of a functional sequence (promoter sequence)
Modified nucleotide that contains labeled group,
biotinlyated, or fluorophore.

9. Cassette
Mutagenesis

Used to introduce
multiple mutations
into the DNA
sequence
Synthesis oligo

T4 lysozyme: a model for stability studies

An example
Cysteines were added to
areas of the protein in
close proximity--disulfide
bridges could form
T4 lysozyme: structure known
Can it be made more stable by
the addition of pairs of cysteine
residues (allowing disulfide
bridges to form?) without altering
activity of the protein?

30-May-16

Stability can be increased - but there can be a cost in activity