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Site Directed Mutagenesis

Site directed mutagenesis is a method used to intentionally alter the DNA sequence of a gene. It works by synthesizing a DNA primer containing the desired mutation that is complementary to the template strand. The primer is then extended by DNA polymerase to generate a new strand bearing the mutation. The parental DNA is digested by the DpnI endonuclease, leaving only the mutated plasmids. These are then transformed into E. coli cells and clones containing the mutation are identified through DNA sequencing. The key advantages are that it ensures only mutated DNA remains without any parental template, preventing unwanted repair processes from reversing the mutation.

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Cady J'adoreDior Rodney
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76 views

Site Directed Mutagenesis

Site directed mutagenesis is a method used to intentionally alter the DNA sequence of a gene. It works by synthesizing a DNA primer containing the desired mutation that is complementary to the template strand. The primer is then extended by DNA polymerase to generate a new strand bearing the mutation. The parental DNA is digested by the DpnI endonuclease, leaving only the mutated plasmids. These are then transformed into E. coli cells and clones containing the mutation are identified through DNA sequencing. The key advantages are that it ensures only mutated DNA remains without any parental template, preventing unwanted repair processes from reversing the mutation.

Uploaded by

Cady J'adoreDior Rodney
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
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Site directed

Mutagenesis
Strategies and Methods
(QuikChange)
Cady Rodney

Introduction
What is Site Directed Mutageneis (SDM)?
A method used to make specific and intentional
changes to the DNA sequence of a gene.

Why do we use SDM?


Investigation of structure (DNA, RNA and proteins)
biological activity (protein function, interaction, cellular regulation)
Protein engineering

Procedure
How does SDM work?
Synthesis

of DNA primer (containing mutation)


complementary to template strand

Primer

extension by DNA polymerase

Introduction

into a host cell as a vector

Cloning
Mutants

selected by DNA sequencing

Procedure
Synthesis of complementary mutagenic primers
DNA

template is denatured by thermal cycling


reactions

Both

primers applied in this method hybridise to


the same region of the DNA template

Mutagenic

primers are annealed to denatured


template DNA.

Procedure
Primer extension by DNA polymerase
Pfu-based

DNA polymerase extends the


mutagenic primer

generates

ds-DNA molecules with one strand


bearing multiple mutations

Nicks

are sealed (ligated) by components in the


enzyme blend.

Procedure
Treatment with the restriction endonuclease Dpn I.
Dpn

I endonuclease (target sequence: 5-Gm6ATC-3) is specific for


methylated and hemimethylated DNA5

used

to digest the parental DNA template

Other SDM methods leave a mixture of wild type (methylated) and mutant
strands (non methylated). The host cell may use the methylated strand as the
template for repair, removing the desired mutation.

Procedure
Finishing up

Transformation
Mutant

into XL10-Gold ultracompetent cells

closed circle ss-DNA converted into duplex form

in vivo
Double

stranded plasmid DNA prepared from


transformants

Analysis

to identify clones bearing desired mutation

Summary

Advantages
Finishing up
No

parental (wildtype ) DNA left

No

mismatched pairs left

No

activation of mismatch pairs

No

activation of mismatched repair

THE
END!

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