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Long PCR Primers? Advice for generating multiple adjacent mutations


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#1 jeremyn

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Posted 25 July 2010 - 11:48 PM

Hi,
I'm trying to use shotgun alanine scanning to test the importance of 7 key residues in a protein. Can anyone give me advice on how best to achieve that many simultaneous, consecutive mutations?

I've made myself a tool to find minimally degenerate codons, but am not sure how I'll actually do the mutagenesis. Would it be foolish to try to design one long PCR primer (following the Megaprimer mutagenesis method) that incorporates all 21 bases? I'm assuming that I'll need at least 20 BP on either side to anchor the primer down. This would mean using a 65mer.

When I analyzed this using the IDT oligo analyzer, it was throwing up a heap of secondary structures and self dimerization. Can anyone give me advice on tools to use when designing such long primers or tell me if there's a better way to do it?

Edited by jeremyn, 26 July 2010 - 02:02 AM.


#2 laurequillo

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Posted 26 July 2010 - 12:44 AM

Hi,
I'm trying to use shotgun alanine scanning to test the importance of 7 key residues in a protein. Can anyone give me advice on how best to achieve that many simultaneous, consecutive mutations?

I've made myself a tool to find minimally degenerate codons, but am not sure how I'll actually do the mutagenesis. Would it be foolish to try to design one long PCR primer (following the Megaprimer mutagenesis method) that incorporates all 21 bases? I'm assuming that I'll need at least 20 BP on either side to anchor the primer down. This would mean using a 65mer.

When I analyzed this using the IDT oligo analyzer, it was throwing up a heap of secondary structures and self dimerization. Can anyone give me advice on tools to use when designing such long primers or tell me if there's a better way to do it?


You could check for companies that produce synthetic genes. I have used some of them and they are quite useful! You just tell them the sequence and then once they produce it you introduce it in your vector (exchange the WT sequence for the mutated one). If you have to create several mutations it is a fast and reliable method, and it is not expensive.
you just have to find the proper restriction sites in your sequence
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#3 jeremyn

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Posted 26 July 2010 - 02:01 AM


Hi,
I'm trying to use shotgun alanine scanning to test the importance of 7 key residues in a protein. Can anyone give me advice on how best to achieve that many simultaneous, consecutive mutations?

I've made myself a tool to find minimally degenerate codons, but am not sure how I'll actually do the mutagenesis. Would it be foolish to try to design one long PCR primer (following the Megaprimer mutagenesis method) that incorporates all 21 bases? I'm assuming that I'll need at least 20 BP on either side to anchor the primer down. This would mean using a 65mer.

When I analyzed this using the IDT oligo analyzer, it was throwing up a heap of secondary structures and self dimerization. Can anyone give me advice on tools to use when designing such long primers or tell me if there's a better way to do it?


You could check for companies that produce synthetic genes. I have used some of them and they are quite useful! You just tell them the sequence and then once they produce it you introduce it in your vector (exchange the WT sequence for the mutated one). If you have to create several mutations it is a fast and reliable method, and it is not expensive.
you just have to find the proper restriction sites in your sequence

Thanks for the reply Laurequillo. Companies like GeneArt do offer site saturation mutagenesis and the cost is coming down, but my supervisor wants to develop a service for researchers on a budget. Maybe in a few years that will be the way forward, but for my project, we want to stick to PCR based mutagenesis.

I've seen quite a few posts from people who have successfully used primers of 100BP or more. I'd like to find out from them whether the online oligo analyzer software programmes predicted unfavourable secondary structures and whether they went ahead anyway.

#4 Rsm

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Posted 26 July 2010 - 04:19 AM

I have used a 98mer and several 60-70mers, no big problem. You might need to play around with DMSO or annealing temp.
However, I would suggest you to use overlap extension PCR. That is reverse primer with 20nt of your gene and then 21nt mutation, and forward primer 21nt mutation and 20nt of your gene. Make first PCR with 5' ATG primer and rev and 3'TAA primer and for, and second PCR (after clean-up) with ATG and TAA primer only. Works better, cheaper and quicker.

cheers,

Minna
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