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Deletion mutagenesis
Posted by: charan (IP Hidden, Junior member, 14)
Date: February 16, 2006 07:48PM
Hi,
I am trying to delete 10 amino acids from the middle of a gene(2240 bps) cloned into a 3000 bps plasmid. Any suggessions as to how to do this? Thanx.
Re: Deletion mutagenesis
Posted by: username132 (IP Hidden, Junior member, 15)
Date: February 19, 2006 07:09AM
The following reference gives details of a technique called overlap extension PCR which I think is what you need.
Higuchi, R., B. Krummel and R. K. Saiki (1988). "A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions." Nucleic Acids Res 16(15): 7351-67. Basically, you need four primers. Two of these are complementary to the opposite ends of your gene. The other two are targetted towards the middle and have bits that match the opposite side of the part that you want removed. I've made a diagram using my mad photoshop skillz to make it clearer Following the diagram, the big green bit is your gene. The black bit is the evil part that you want removing. You need to set up two PCR reactions using primers which are split into two regions. One region is complementary to the part to which it is meant to bind, the other part contains say five nucleotides from the sequence of your gene AFTER the evil bit. You need primers (not shown in my diagram) to bind the strands you've just synthesised so you can amplify them as per standard PCR. Then you mix the products of your two reactions, denature and allow them two reaneal. They can reaneal in one of three conformation; a) how they were before you denatured them, b) with overlap at the 3' ends c) with overlap at the 5' ends. Your polymerase will then extend the sequences from the complexes with 3' end overlap. Denature, reaneal, extension a few times and most of the strands will now be full length. Amplify your full length strands with the outermost of your four primers and then gel purify and do what you want with it... what do you want to do with it? The only thing I'm yet to figure out, is how oligonucleotides are synthesised (I'm just a lowly student and only know about OE-PCR because it's in my dissertation) such as to produce for example "VNS" sequences (where V = adenine, cytosine, or guanine; N = adenine, cytosine, guanine, or thymine; and S = cytosine or guanine). I'd imagine adenine for example. left over from the V and N steps would contaminate the S step. I think the process is called "codon doping" but I can't find any information on how it's done. If you could point me in the right direction I'd appreciate it! Edited 4 times. Last edit at 02/19/06 07:42AM by username132.
Re: Deletion mutagenesis
Posted by: username132 (IP Hidden, Junior member, 15)
Date: February 19, 2006 07:09AM
[[my bad]]
Edited 1 times. Last edit at 02/19/06 07:35AM by username132.
Re: Deletion mutagenesis
Posted by: username132 (IP Hidden, Junior member, 15)
Date: February 19, 2006 07:34AM
[[my bad II]]
Edited 1 times. Last edit at 02/19/06 07:35AM by username132.
Re: Deletion mutagenesis
Posted by: charan (IP Hidden, Junior member, 14)
Date: February 19, 2006 11:32AM
Hi,
Thanx a ton. Iwas thinking of this method as I was posting the original message. Your diagram made it so much more easier to figure out the exact strategy. Just one question. Do you directly clone your final product into the expression vector or do you carry out an extra step of cloning into cloning vector like T vector etc.? Thanx once again.
Re: Deletion mutagenesis
Posted by: username132 (IP Hidden, Junior member, 15)
Date: February 19, 2006 04:40PM
No problem! I think it's wicked I was able to help a *real* scientist with a *real* experiment! Unfortunately, I'm not so hot on the second question. If your gene has restriction sites compatible with your chosen expression vector, then I don't see any reason why you can't just put it straight into there. But I am just a uni student and I've never done anything like that before!
It would be cool if you came back to let me know how you got on!
Re: Deletion mutagenesis
Posted by: Basshead (IP Hidden, New member, 4)
Date: February 22, 2006 07:24AM
That's good advice. I can definately reccomend overlap extension as a mutagenesis method it's worked well for me in the past.
For many people cloning the mutant PCR product is the step that they fall down at - when I have a student who has problems with the cloning step I get them to use the Promega pGem T-Easy system and to transform Stratagene's Ultracompetent bugs. This is not the cheapest option but it alweays works. It does require that you tail the ends with Taq Pol (if you haven't used it for the PCR) and you may have to subclone again for expression etc. B
Re: Deletion mutagenesis
Posted by: charan (IP Hidden, Junior member, 14)
Date: February 24, 2006 12:12PM
Thanx all of you who bothered to see and comment. I agree that OE PCR is the ideal technique for this kind of mutagenesis, but we came up with an alternate strategy. There is a unique Resriction site just before the region to be deleted. I am planning to introduce another site for the same enzyme at the end of the region to be deleted by quick change. Once this is accomplished, I will digest with this enzyme , ligate and hopefully get the portion out (unless I have some technical issues with the mutagenesis and/or digestion/ligation). Can you think of any flaw in this strategy?
Re: Deletion mutagenesis
Posted by: username132 (IP Hidden, Junior member, 15)
Date: February 26, 2006 08:05PM
I can't help with this, but have some questions of my own! I was wondering how you would introduce another restriction site into the sequence? Also, since I hope to be doing research (therapeutic gene modulation would be great) within a year, a) what would be the cost of you trying out your idea and failing and b) when you have a problem (such as a question of technique) what do you do whilst you're waiting for the answer?
Re: Deletion mutagenesis
Posted by: charan (IP Hidden, Junior member, 14)
Date: February 27, 2006 12:25PM
Hmm, a question in reply to a question. No problem. ........
The answer to your question as to how to introduce this change is Quick Change mutagenesis(stratagene kit QC). I plan to introduce a RS using QC mutagenesis wherein by changing a couple bases in a single oligo, I guess at least theoretically, I can introduce a new site. I dont quite understand what do you mean by the cost of trying and failing. But if you mean to ask what backup I have, the answer is yes,I do have a scheme to fall back on in case things dont work as expected. f you carefully look at the oligo design for an OE PCR, the inner mutagenic primers can be the same as the ones for QC. So if QC does not work , I can always design the outside primers and carry out and OE PCR. It pays to toy around with a couple different things at the same time, and when I have a problem, I guess I would write to you guys :)
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