Transcript Apr. 5 Presentation Mutagenesis Methods

Mutagenesis Methods
Lily Peterson
April 5th, 2010
Overview: Types of Mutagenesis
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PCR Based Methods
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Insertional Mutagenesis
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Site-directed mutagenesis
Mismatched mutagenesis
5’ add on mutagenesis
Cassette Mutagenesis
Trasposon mutagenesis
In vivo Mutagenesis
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Direct Mutagenesis
Mismatched Mutagenesis
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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.
Gene or cDNA
is cloned into
M13 vector
Use of M13 allows for single
strand recombinant DNA
recovery
Screen for
mutant
5’ add on Mutagenesis
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Involves adding on a new sequence or
chemical group to the 5’end of a PCR product
This involves a particular way of designing
the primers:
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3’ end of the primer matches the sequence of
PCR product.
5’ end contains the novel sequence
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Suitable restriction site
Addition of a functional sequence (promoter sequence)
Modified nucleotide that contains labeled group,
biotinlyated, or fluorophore.
Uses and Limitations
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PCR based methods
are useful in making
specific mutations in
the DNA
Which is useful when
studying different
aspects of protein
function
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With PCR based
methods it is hard to
replicate the mutated
DNA…in order for
replication to occur
super competent cells
must be used and are
expensive!
Screening can be
tedious, usually
requires sequencing to
confirm if mutation
occurred.
Cassette Mutagenesis
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Used to introduce multiple mutations into the
DNA sequence
Uses blunt ended DNA for insertion site of
mutation
Where mutation is inserted a 3 base pair
direct terminal repeat is created
The mutagenic codon cassette has two head
to head SapI sites allowing for removal of all
DNA except for mutation.
Targeted codon removed
using restriction enzyme
that creates blunt cut
SapI digestion
creates 3’
overhang allowing
for ligation.
Ligation, which creates final
mutation
Uses and Limitations
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Typically used for
protein structure but
possibly used for gene
function
Less expensive than
site directed
mutagenesis to create
several mutations,
because there is no
need for primers
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Requires the SapI
restriction enzyme cut
sites, and other cut
sites flanking the target
region for removal of
DNA
Works best when target
region is contained in a
small DNA fragment
Transposon Mutagenesis
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Transposon: a piece of short DNA that replicates by inserting into
other pieces of DNA (plasmids, chromosomes, etc…)
Useful for studying gene function because when the transposon
moves into different location in the DNA it may cause a disruption
in a gene or a set of genes.
Transposons also have many useful properties for mutagenesis:
 Cause clean mutations
 Can be random or specific mutations
 Typically encode for antibiotic resistance or some other
advantageous gene.
 Can use a transposon that inserts at a high frequency
 When used in bacteria it causes selectable phenotypes
 Recognize specific sequence that is ~2-12 base pairs long
Uses and Limitations
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Primary use is for the study
of gene function, though
can be used to create gene
fusions
Usually easy to see a
change in phenotype due to
gene knockout
Because the transposon
inserts at a specific
sequence, helps in
determining where insertion
occurred
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Not useful in large plasmids
because many recognition
sites could be contained in
the a single plasmid
Suicide vectors are used,
though some may have
limited replication, so further
screening is needed
Sources