Inducing UV mutations in a gene

[Ivaylo]

Hello!! I got a problem and the whole thing is a mess. I want to induce UV mutations in a gene and see how this protein is gonna work. How is this thing done?? How should I effect only the gene I am interested in??? Thanks !!!

[ecoli]

if you're planning on subjecting bacteria to UV light, it will be impossible to limit mutations to a single gene, at least, AFAIK.

 

You can still test the sequence of a desired gene, using the sanger method. Although I don't know if gene sequencing is an option for you. Are you working in a university setting?

[Ivaylo]

If I induce a mutation in a gene, I can isolate the protein, but I want to see the mechanism this mutation will effect, so it should be in a living cell. I can use PCR and then expose to UV - that's how I can get a mutated gene, but how to place it in a living organism so I can see what is gonna happen? Mind my dumb questions, but I am learning now, I would really appreciate anw answers.

 

P.S Is there any mechanism so I can mutate (let's say) a specific nucleotide in a gene - a selective mutation of some sort???

[Ivaylo]

Ecoli,

Yes, I am working in the university.

[CharonY]

As already mentioned, UV mutagenesis is highly unspecific. It randomly introduces thymidin dimers and you will be at a loss to see where (if at all) a mutation happens. Sequencing won't help you much here, either.

However, there are a lot of cloning techniques which allow a specific mutagenesis of genes with known sequences. The simplest one is disruption of a gene by integrating a vector into it. In short:

- generate internal fragment of the gene by PCR

-clone that PCR fragment into a suitable vector that is not replicated in the organism in question (suicide vector). This vector should be selectable (e.g. by ABs)

-introduce the vector in organism in question and select for the vector. Those cells which get to be selected possess the vector integrated into their genome (via homologous recombination over the internal fragments)

 

The result is usually a truncated protein that is usually quickly degraded.

Similar techniques can be used to create fusions or even base specific mutations. For these you need two recombination events, though (homogenotisation).

 

For more on that I would recommend some text books. One of the most often used ones is Sambrook and Russel: Molecular cloning.

Check it out in the library it is a very useful read.

[Dak]

couldn't you put the gene into a plasmid, UV mutate it, and then plonk the plasmid into the bacteria?

 

that way, only the gene of interest would be mutated.

[CharonY]

Well, if you use a plasmid in the first place using site specific mutatgenesis would be far more time efficient. The mutation has to be by chance within the gene and the vector should not be mutagenized (at least not in a negative way). For this you would have to screen all the plasmids in the hope of finding one with a mutagenized insert.

 

Also if you want to analyze the resulting protein, you would have to express the protein heterogenously e.g. using a expression vector. If the UV mutagenizes the vector instead of the insert (which is very likely as most expression vectors are larger than a single gene) it might not work at all.

 

Essentially it would be much faster to produce a mutagenized fragment via PCR and then clone that into a vector. You'll have to amplify the gene to clone it, anyway.

[Bluenoise]

couldn't you put the gene into a plasmid, UV mutate it, and then plonk the plasmid into the bacteria?

 

that way, only the gene of interest would be mutated.

 

Sure but you'd still have a working copy on the chromosome.

 

I'm having trouble seeing what exactley is the goal here. If you just want to knock out a particular gene well I'd go about it as CharonY suggested. Unless it's an essential gene, things get a little more complicated then.

 

If you want to look at the effect of specific mutations, then knock it out and complimet it with mutatated versions on plasmids.

[Dak]

sorry, yeah i meant knock it out and replace it with a mutated version on a plasmid.

 

but like charon said, putting it on a plasmid then mutating it (my original suggestion) isn't a good way of doing it

[Ivaylo]

Ok, I will try to do it that way with cloning and selective mutations and I will try with UV light cause I am curious what's gonna happen. Thanks everybody for the discussion and special thanks to CharonY. I will try to find the book, it seems I'll need it.

[lucaspa]

P.S Is there any mechanism so I can mutate (let's say) a specific nucleotide in a gene - a selective mutation of some sort???

 

Yes. Do a PubMed or Google search on "site specific mutation".

[Ivaylo]

http://www.madsci.org/posts/archives/oct2001/1004108157.Mb.r.html

Here I found in brief some useful information for the site specific mutagenesis. For more information, type "site specific mutagenesis" in google.

[Prewitt]

i agree with the above. best would be to have a strain without the protein (then you can see what it does anyway, there are better means for that) and then put a plasmid with the chormome in it. you have to show that these are equal if you want to make conclusions about in vivo. then you can make a strain with a mutated plasmid and can compare these. i'd suggest in vitro studies before that though. translate it in vitro translations n stuff. better to start basic that complex.

[lucaspa]

http://www.madsci.org/posts/archives/oct2001/1004108157.Mb.r.html

Here I found in brief some useful information for the site specific mutagenesis. For more information, type "site specific mutagenesis" in google.

Why should I do this? YOU are the one that needed the info. All I have to do is refer to my molecular biology textbook or walk down the hall and talk to my colleague who does this routinely.

[HuskerNation]

Just another thought on this, considering my master's thesis is on UV mutagensis in marine bacteria...

 

You will want to use germicidal (UV-C) which is in the range of 190-290nm. This is the type of damage which causes direct damage (pyrimidine dimers, 6-4 photoproducts, rare base adducts, etc.). If you use UV-A (320-400nm), you will only get indirect damage via reactive oxygen species, and not even necessarily in the DNA, but also could effect enzymes and other proteins.