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What is site-directed mutagenesis?

The intentional mutation of a specific site within a DNA sequence

It is important because it gives you control
It allows you to ask specific questions about the importance of a DNA sequence within the context of your experimental system


What information is needed for site-directed mutagenesis of protein coding DNA?

DNA sequence of the protein coding gene
3D structure of the protein from X-ray crystallography, NMR, or cryoEM
Information about its ‘active site’
An activity assay to evaluate mutant enzymes in comparison to wild-type


What are some examples of types of site directed mutagenesis?

Changes to non-protein coding DNA:
You may need to mutate out an inconvenient restriction site in the middle of your gene of interest so you can clone it via restriction enzyme cloning
You may want to investigate the importance of a specific nucleotide(s) within: promoter regions, eukaryotic splice sites or RNA secondary structure

Changes to protein coding DNA:
You may want to better understand the contribution of a particular amino acid residue to the 3D structure of a protein and/or its functionality
This is probably the most common use of site-directed mutagenesis


How is site directed mutagenesis carried out in general?

Select gene of interest
Identify the amino acid residue of interest
Then 2 options:
Generate SDM using PCR methods
Pay a company to synthesise mutant DNA(s) for you

Then complete your experiment


What can you buy from a company - that synthesise mutant DNA for you?

IDT gBlock
An IDT gBlock is a linear dsDNA molecule
It is typically used directly for Gibson assembly or as a PCR template for conventional restriction enzyme cloning
You can also pay the company to clone the DNA for you, but this is more expensive - hundreds to thousands of pounds


How do you design a gBlock that habours a point mutation?

You find an essential active site residue
Change this
Copy/paste into the order form for a company and purchase it


What are some tips for working with DNA?

When typing DNA sequences use Courier New font
No matter what the character is, it will occupy the same space, so sequences will line up nicely

It is uncommon for DNAs to be written as dsDNA, so you need to get comfortable with seeing/working with ssDNA, typically the ‘coding strand’

It is ‘convention’ to write DNA molecules in a 5´-to-3´ orientation (directionality of DNA synthesis)


What are the PCR methods you can use to generate site-directed mutations?

QuikChange Mutagenesis
Enzymatic Inverse PCR (EIPCR)


Describe the QuikChange mutagenesis?

1. Clone your gene of interest into your plasmid or vector of choice
2. Design mutagenic primers that are complementary to one another and harbour the desired mutation
3. Perform a PCR reaction with the mutation-containing primers and the plasmid using a high-fidelity polymerase
This results in nicked circular strands
4. Treat the QuikChange reaction with DpnI restriction enzyme to destroy methylated template plasmid
5. Transform E. coli
6. Screen transformants, and sequence verify the mutation


Describe what happen during the QuikChange mutagenesis PCR?

Heat dsDNA into ssDNA
Primer annealing - mismatch primer annealing between the mutagenic area
DNA synthesis - the primer is extended in to 5' to 3' direction (around the circular plasmid)

After one round:
We have the original and mutant codon = hybrid
There is a nick - a phosphodiester bond is not created between a GG
GG - The last base added in DNA synthesis and the base of the primer

Subsequent rounds:
When DNA is denatured, because there is a dsDNA 'nick' this prevents the DNA polymerase from extending the primer
Therefore this is a linear amplification (not exponential like most)
Only the original plasmid can serve as a PCR template
This PCR reaction is very inefficient


What happens in the final stage of QuikChange mutagenesis PCR?

If you transformed E. coli with your QuikChange reaction now = a lot of false positives - the original template plasmid will transform much more efficiently than nicked circular strands

The QuikChange reaction is digests with the DpnI restriction enzyme
DnpI - digests methylated DNA
Our plasmid is methylated

1. Use the DpnI-digested QuikChange mix to transform E. coli
2. Select a few colonies to inoculate overnight cultures
3. Mini-prep the overnight cultures to obtain purified plasmid DNA
4. DNA sequence to confirm that the intended mutation was generated


Describe the enzymatic inverse PCR (EIPCR)?

1. Clone your gene of interest into your plasmid or vector of choice
2. Design a set of mutagenic primers with restriction enzyme sites at their 5’ ends
3. Perform a PCR reaction with the primers and the plasmid using a high-fidelity polymerase
4. Digest the PCR product with the restriction enzyme from Step 2 and DpnI
5. Perform a ligation reaction and transform E. coli
6. Screen transformants, and sequence verify the mutation


What are the rules for EIPCR primer designs?

Primers should anneal back-to-back to the template
Primers should normally be between 15nt to 40nt long
At least 10-15nt of the primer should anneal to the template
Only one of the primers harbours the mutation


What restriction enzymes does EIPCR use?

Type IIs restriction enzymes are used (like golden gate cloning)
It cuts outside of the site it recognises - therefore scarless cloning

When incorporating restriction enzyme sites into primers, you should know that many enzymes do not cut efficiently if the site is right at the end of the primer
Therefore, six extra nucleotides are often included to enable efficient cleavage


Describe the mechanism of EIPCR?

dsDNA denaturation to ssDNA
Primer annealing for EIPCR - back to back
(reverse primer contains an extra GAGCA for scarless programming)

First round - There are two products and only product two has the mutation

Subsequent rounds:
Primers can extend the original plasmid template AND the newly synthesised DNA, resulting in EXPONENTIAL amplification


What are the four possible outcomes of the subsequent rounds of EIPCR?

Reverse primer + product one
Forward primer + product two
This is effectively amplifying the original template

Forward primer + product one
Reverse primer + product two
We are the most interested in the last one as it is the only one that contains the mutation on both strands and have two BsaI sites


What is left/happens at the end of EIPCR?

There will be a mixture of products in the reaction
The one that we want is digested with BsaI
This can be done due to the extra portion of DNA that allows scarless insertion
We do a standard T4 DNA ligase, transform E.coli and screen/sequence to confirm the mutation


What are other concepts relating to site directed mutagenesis?

Alanine Scanning
Site-saturation mutagenesis


Describe Alanine screening?

Using site-directed mutagenesis to alter the wild-type codon to one specifying alanine to determine the contribution of the original residue to the stability or function of the protein
Important for defining/confirming the active site of an enzyme you are studying

Alanine is often used because its ‘R group’ is simply a methyl (not involved in catalysis or binding)


What is site-saturated mutagenesis?

Systematic mutagenesis of the wild-type codon to codons specifying the remaining the amino acids

To do the experiment:
Choose to use QuikChange or EIPCR
Design 19 sets of primers
Perform the reactions


Give an example of site-saturated mutagenesis?

Active site of galactose oxidase

Create 19 mutant variants where W290 was changed to another amino acid
Purify resulting proteins
Compare enzyme performance using an activity assay

All mutations killed catalysis except for two
Calculate Km and Kcat
The lower the Km = higher affinity for the substrate
Kcat - is the turnover number
Therefore they can observe how the active site is affected by altering an amino acid residue

The tryptophan was involved in orientation of substrate binding so the copper ion could do the catalysis


What is used to improve function of proteins?

Directed Evolution

It is difficult to predict changes to improve function because we do not really understand how proteins work
Several amino acid changes are often required together to improve function, and these are difficult to rationally identify so therefore directed evolution (random mutagenesis/iterative screening) is used instead