How to construct a Double mutant - (Feb/20/2008 )

Hi,

I have two single mutants of a protein and I want to construct a double mutant using these two. How should I go about?

Thanks

take one of the single mutants and make the second mutation in it.

Sorry to say this, but I found that reply very amusing.

You can also take the plasmid expressing the first mutant protein and cut out part of the gene containing the mutation. You then take the plasmid of the second mutant protein , remove the same session (which is normal in this plasmid) and replace it with the mutant session. Ligate the pieces together and you will get your double mutant.

Thank you so much. I could not understand the word mutant session. I want to use PCR in this construction. What primers I should use?

Thanks again

section... spelling mistake. i ment the section of the gene containg the mutation.

Using PCR?

Well, there is site directed mutagenesis. As Lotus mentioned, using site directed mutagenesis you make a second mutation to gene already containing a mutation.


The second method using PCR that I can think off, uses two set of primers pairs to amplify the gene in parts, introducing the mutation as part of the primers. Both halves are then fused together either by PCR or SLIC.

Firstly you have to make 2 sets of primer pairs. Primer pair 1 and 2, primer pair 3 and 4.
The template for both PCR reactions contains the first mutation.
Primer 2 and 3 both introduce the second mutation. Both primers have about 25bp to 30bp of overlapping sequence.
Primer 1 and 4 should contain unique restriction sites that allow you to clone the finished product into your expression vector.
All four primers must have the same melting temperature. I would recommed a melting temperature of 58 to 60 Celsius. Only the part of the primer that binds to the template initially is considered to make the tm calculations.

=Method=
Firstly a PCR reaction is conducted using primer pair 1 and 2, producing PCR product A
Next a second PCR reaction is conducted using primer pair 3 and 4, producing PCR product B.

At this point one may use PCR to fuse product A and B together.
Add equal quantities of A and B together (~20ul +20ul) . Add dNTP and PCR buffer and polymerase.
Melt the two products and reannealing them together. Run sufficiently long extention time for the polymerase fills in the missing bp. This reaction is not PCR.... there is no cycling. Think of it instead as a megafill in reaction.

You may then clean up the reaction, restriction digest and clone the fused product into your vector, should the concentration of fused product be sufficient. Alternatively you may use primer 1 and 4 to amplify the fused product by PCR.

If desired you may used SLIC...a recombination method to fuse DNA fragments with sequence (30bp) homology using recA and T4 DNA polymerase. Unfortunately I can not vouch for this method as I have not done it before

Thanks for your suggestions. As per site directed mutagenesis, I am thinking instead of 4 primers, I will use only two. The two primers will have the second or desired mutation that will be introduced into the first plasmid (containing first mutation) via thermal cycling (PCR using pfu). This will give palsmid with both mutations and also plasmids with no mutations (these plasmids can digested with DpnI enzyme). The plasmid with both mutations will be transformed without any second PCR. Do you think this will work?

Thanks

Site directed mutagenesis should work it is a standard method. You should check google for more information on the protocol. You need to use DpnI to digest/remove the old (melthylated) copy of the plasmid.

Thank you so much. I am now clear about this experiment.