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Better option, mutagenesis or gene synthesis?


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#1 HOYAJM

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Posted 24 October 2012 - 09:15 PM

Hi, I have a 5kb sequence that I need to change, twice. Each construct will have 6 amino acids changed (they are all back-to-back). I have done a bit of site-directed mutagenesis, but only to change 1 amino acid at a time. Since I have to change 12 amino acids between two constructs, I am wondering if this feasible, in a acceptable amount of time.

I am considering gene synthesis, but for the 5kb sequence, it would be upwards of $2000 each. So I would have a ~500bp sequence synthesized and then subclone it into my vector that I already have. This would cost ~$300-400.

My question is, is it possible to change 6 amino acids in a row with site-directed mutagenesis in a reasonable amount of time? Unless I can change 2-3AA at a time, I think this would be a lengthy undertaking. Does anyone has information on SDM that can mutate multiple residues at once?

If I am totally ignoring other possibilities, please share.

Thanks

#2 Curtis

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Posted 25 October 2012 - 12:54 AM

I have done site-directed mutagenesis before but only on 1 aa. I understand why you are in doubt. 6 aa difference is quite big. I am also not sure if it will amplify.

gene synthesis is the easier option but it's obviously costlier. However I think $2000 is quite cheap. This company called DNA 2.0 in California synthesizes large fragments for $1.25 per bp.

#3 GNANA

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Posted 25 October 2012 - 02:52 AM

As Curtis said gene synthesis would be the appropriate option to chang 6 amino acids residing back to back, but you can still opt SDM if you can alter atleast two aa, for example, if altering just the last nucleotide of first and any of first 2 of the second aa could change both in a single round of PCR !!! obviously you then do it sequentially keeping the altered construct as the template.
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#4 HOYAJM

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Posted 26 October 2012 - 06:43 AM

I have done site-directed mutagenesis before but only on 1 aa. I understand why you are in doubt. 6 aa difference is quite big. I am also not sure if it will amplify.

gene synthesis is the easier option but it's obviously costlier. However I think $2000 is quite cheap. This company called DNA 2.0 in California synthesizes large fragments for $1.25 per bp.

As Curtis said gene synthesis would be the appropriate option to chang 6 amino acids residing back to back, but you can still opt SDM if you can alter atleast two aa, for example, if altering just the last nucleotide of first and any of first 2 of the second aa could change both in a single round of PCR !!! obviously you then do it sequentially keeping the altered construct as the template.


Curtis, if you are ever interested in gene synthesis, check out GENEWIZ, GeneART, GenScript, as they are all under $0.50/bp, as low as $0.35/bp for large fragments.

GNANA, I will definately have to look at how many nucleotides I would have to change before I decide, you could be right and I wouldnt have to change 10+ nucleotides if I get lucky.

Thanks

#5 Curtis

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Posted 26 October 2012 - 11:44 AM

OK, thanks




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