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I'm looking to generation sequential deletions from a gene of interest. The total size of this region is 8 amino acids. I'm trying to determine which portion of this region is necessary within the gene.

For example, if this is my sequence:
ABCDEFGH

I want to generate:
BCDEFGH, CDEFGH, DEFGH, and so on.

What is the least expensive way to do this (by minimizing primer use, enzyme use, etc). I already have this gene sub-cloned into an expression vector.

Thanks!

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How big is your gene and your vector? –  Gergana Vandova Feb 29 '12 at 4:39
    
The gene is 600 bp and vector ~5 kb. My thinking is that I would need a whole new primer set for each amino acid deletion. –  leonardo Feb 29 '12 at 18:40
    
I'll consider this question answered. I gave @bobtejoe best answer because he broke down the costs and a suitable method for making the sequential deletions. Amy gave useful considerations for using conservative mutations over deletions to preserve protein conformation and possibly function. QuickChange is not the cheapest kit to buy, but the costs seem to be minimized when OE-PCR is used in conjunction with homemade stocks of reagents. –  leonardo Feb 29 '12 at 22:03
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2 Answers 2

up vote 6 down vote accepted

While it isn't the cheapest, it is certainly the fastest and simplest. I would quikchange out the amino acid. This would require no subcloning and only require

  • two ~25 nt primers ($10)
  • 1 shot of pfu (~$0.25)
  • 1 shot of DPNI ($0.05)
  • competent cells (~$5)
  • sequencing to confirm (~$4-6)

Overall, probably >$20 a mutant all in 2-3 days of waiting.

(edit) I'm going with quikchange rather than OE-PCR simply because it removes the cloning steps. You will potentially need to screen more colonies with conventional cloning vs. quikchange.

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How would you quikchange out the last amino acid? Replacing with a stop codon? –  Gianpaolo R Feb 29 '12 at 8:49
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If we're thinking about this problem from the context of traditional biochemistry, I would just have nothing. That is if your gene is XABCDEFGHY, you now have XY. You still have the context of the rest of the "gene" to observe. It should be a nice negative control. –  bobthejoe Feb 29 '12 at 9:30
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@leonardo the cheapest competent cells are the ones you make yourself! –  Amy Feb 29 '12 at 19:27
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I've always made my own competent cells (DH5alpha cells which is why I don't know the costs). I would assume that you would want to keep your vector in an expression strain so if you want to skip a step, I would transform straight into BL21. –  bobthejoe Feb 29 '12 at 19:49
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If you are going to clone directly into BL21 - use one of the "gold" strains so you can pull the plasmid out later with less chance of random mutations. –  Amy Feb 29 '12 at 21:11
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Deletions may make sense if you are analyzing the N-terminus or C-terminus of a protein. If you are looking at an internal region however, keep in mind that the more AAs you delete, the more likely you are to disrupt the overall protein structure. If you delete any random selection of 8 AAs within a protein, there's a chance you'll knock out activity by changing the protein fold or stability. That's not useful information.

This question is usually first addressed by alanine scanning - sequentially or additively changing amino acids to alanine. This is much more informative than deletions. Even better, you can choose to replace wild-type AAs with other AAs of similar size but differing charge or hydrophobicity. Then you are most likely to change the function of a region without changing the structure.

The quickchange kit works great, but if cost is an issue you can do whole plasmid mutagenesis PCR with your own reagents. And make your own competent cells.

In my experience though, whole plasmid PCR can be tricky - if it doesn't work the first time it can be difficult to troubleshoot. If time is an issue, I'd recommend doing overlap extension PCR with the same mutagenic primers, plus one set of amplification primers at the 5' and 3' end of your gene. Make a large batch of digested vector, test as a negative control, and use it for ligation of all of your different inserts.

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I recently made 6 different constructs (single and double mutants) using overlap extension PCR. It took me one week, including sequence verification AND was a fraction of the cost for a quickchange kit. –  Amy Feb 29 '12 at 19:51
    
OE-PCR might be a good way to go too. I'll have to work out the primers that I would need. I'm aware of the uses of alanine scanning and conservative substitutions, but for my purposes these amino acids are in an unstructured region, at the C-terminus and deletion will tell me more information about the localization of the protein rather than function. –  leonardo Feb 29 '12 at 20:30
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I may be biased but there is an entire MATLAB toolkit developed for this exact purpose for highthroughput alanine scanning using overlap extension. The nice benefit is that your costs gets reduced to only 1 oligo rather than 2 oligo per mutant. simtk.org/home/na_thermo –  bobthejoe Feb 29 '12 at 20:38
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@leonardo that makes much more sense if you're making truncations at one end. I'll add that to my answer so it is more broad. –  Amy Feb 29 '12 at 21:07
    
@bobthejoe - wow I didn't know about that! I've been designing them by hand like a sucker. Thanks. –  leonardo Feb 29 '12 at 21:59
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