How to detect DNA mutations in bacterial cells

[pine_smile]

Dear everyone,

 

I have basic a question about detecting DNA mutations in bacteria. I am investgating whether bacteria (P. aeruginosa) can develop resistance to UVC irradiation . If resistance develops, my hypothesis is that DNA mutation is induced by UVC irradiation. How to prove this? Sorry for this basic question, becuase I am an bioengineer and DNA questions are out of my field.

 

Thank so much!

[Paralith]

Dear everyone,

 

I have basic a question about detecting DNA mutations in bacteria. I am investgating whether bacteria (P. aeruginosa) can develop resistance to UVC irradiation . If resistance develops, my hypothesis is that DNA mutation is induced by UVC irradiation. How to prove this? Sorry for this basic question, becuase I am an bioengineer and DNA questions are out of my field.

 

Thank so much!

 

Well, unless you're looking for mutations in a specific gene, you're going to have to sequence the whole genome of your bacteria before and after UVC irradation and compare the two. You'll probably also have to do some calculation to take into account the natural rate of mutation as well.

[pine_smile]

Dear Paralith,

 

Thank you very much for kind response. It's really helpful. Could you recommend to me any written materials (e.g. publications) on how to perform the sequencing of the whole genome of the bacteria?

 

Thanks again!

 

 

 

 

 

Well, unless you're looking for mutations in a specific gene, you're going to have to sequence the whole genome of your bacteria before and after UVC irradation and compare the two. You'll probably also have to do some calculation to take into account the natural rate of mutation as well.

[Paralith]

Not of the top of my head, and rather than have me search for you, head on over to this web site:

 

http://www.ncbi.nlm.nih.gov/sites/entrez

 

That should help you find some good papers. I'm sure there are lots from the people who did the human genome project and things like that. I usually work with specific genes, myself.

[ecoli]

If your looking for general sequencing techniques, the most commonly used today in the Sanger method. A google search will give you some good introductory resources.

 

Also, if you look through the sie posted by paralith above, you can find a databse of fully and partially sequenced organisms (a lot of different bacteria species).

[pine_smile]

Thanks, Paralith!

Thanks, Ecoli!

[DrDNA]

I am investgating whether bacteria (P. aeruginosa) can develop resistance to UVC irradiation . If resistance develops, my hypothesis is that DNA mutation is induced by UVC irradiation

I'm unclear on how resistance to UVC postively would correlate with mutation induced by UVC. It seems like the opposite would be true. Maybe I'm missing something.

[pine_smile]

Dear Dr. DNA,

 

Do you mean the mutations will make the bacteria more sensitive to UVC irradiation? If it is this case, what might be the reason for indcuing the resistance? Please advise. Thanks so much!

 

I'm unclear on how resistance to UVC postively would correlate with mutation induced by UVC. It seems like the opposite would be true. Maybe I'm missing something.

[DrDNA]

No. I think I understand what it is that you are looking for now. It was just unclear to me but your statment clarified it.

[Tearfeather]

Dear Dr. DNA,

Do you mean the mutations will make the bacteria more sensitive to UVC irradiation? If it is this case, what might be the reason for indcuing the resistance? Please advise. Thanks so much!

 

Why you think UVC might induce mutations. the survived bacteria as far as I know was mutated that's right, but that doesn't mean mutations were induced in them?!

or maybe I don't understand something.

[ecoli]

Here's another tool that could help you...

 

After your done sequencing, you're going to have to compare your sequence to the published sequence. This website can basically do that for you: http://www.ncbi.nlm.nih.gov/BLAST/

 

Basically it matches up the sequences (it can do it in backwards order to, if you've used reverse primers to sequence, etc).

 

An important thing to remember though when you're doing sequencing, sequencing gets more accurate as you move to the middle of your targeted region. Therefore, if it looks like you see a mutation, esp. towards the beginning or end of your sequence, don't immediately conclude a mutation has occured. You must use primers that overlap sequencing sections and compare those to make sure you eliminate sequencing errors.

 

From your post, it doesn't seem as if you're up to that point yet... but it's something to keep in mind.

[pine_smile]

Dear Ecoli,

 

I really appreciate your comments and suggestions.

 

Here's another tool that could help you...

 

After your done sequencing, you're going to have to compare your sequence to the published sequence. This website can basically do that for you: http://www.ncbi.nlm.nih.gov/BLAST/

 

Basically it matches up the sequences (it can do it in backwards order to, if you've used reverse primers to sequence, etc).

 

An important thing to remember though when you're doing sequencing, sequencing gets more accurate as you move to the middle of your targeted region. Therefore, if it looks like you see a mutation, esp. towards the beginning or end of your sequence, don't immediately conclude a mutation has occured. You must use primers that overlap sequencing sections and compare those to make sure you eliminate sequencing errors.

 

From your post, it doesn't seem as if you're up to that point yet... but it's something to keep in mind.

[DrDNA]

If all you want to do is see if the UVC casues mutation, I'm pretty sure that it would be much easier to expose bacteria to UVC and grow on selective media, for eg, in the presence of an antibiotic (eg, Strep). You would then compare these to control bacteria which are not exposed and calculate the difference in the number of mutant colonies. I recall that we analyzed potential mutagens this way in my undergrad bacteriology lab too many years ago to count....

 

For example:

"This experiment introduces the simple technique of direct selection for the detection and isolation of certain mutants. This technique involves plating bacteria on a selective medium on which only the desired mutants can grow. Specifically, the experiment involves taking a culture of microbes at a high density and plating them on a rich medium containing the antibiotic streptomycin. Streptomycin inhibits protein synthesis by binding to the small subunit of the ribosome and blocking entrance of initiator N-formyl methionine tRNA into the ribosome, thus preventing the start of protein synthesis. A single mutation in the S12 protein of the small subunit of the ribosome prevents streptomycin from binding, thus causing the microbe to become resistant to the antibiotic. By plating a strain onto a medium containing streptomycin, it is possible to fish out the microbes present that have a mutation in the S12 gene. Please appreciate the power of this selective technique. By performing a very simple experiment, it is possible to fish out the 100 or so cells, in a mixture containing billions, that have a change in a specific gene. Also, note how easy it is for a microbe to become resistant to an antibiotic. While the drug is killing 99.9999% of the microbes present, 0.0001% are able to survive, and these microbes are now resistant to the antibiotic. This explains why drug resistance in microbes can occur so rapidly and is a constant problem in medicine."

 

http://www.bact.wisc.edu/microtextbook/index.php?module=Book&func=displayarticle&art_id=127

[CharonY]

I am still not quite clear what the objective of the experiment is supposed to be.

Do you think that radiation might cause mutations that in turn increase resistance to UV radiations? Improbable, but not impossible. However you will only end up (if you are lucky) with UV resistant strains. To demonstrate which mutations were caused is not possible within a normal project budget though (less if it is some kind of course work or undergrad project).

 

Let's go to the suggestions:

- Sequence the genome: in theory it would work, but the price tag and effort to sequence even a small bacterial genome is enormous with traditional Sanger. Newer parallel sequencing methods based e.g. on pyrosequencing are cheaper, but still cost around 15-20k$ per single run. That's about the budget for a phd student for a year.

-use BLAST: BLAST is basically a quick global alignment program used to identify homologous genes/proteins. It is unsuited to detect point mutations (the most likely mutations caused by UV radiation). But regardless, as you got no idea what is going to be mutated you either have to sequence the whole genome, or pick up potential genes involved in UV protection (the P. aeruginosa genome is published), make PCR and check the products for mutations.

- demonstrate that UV causes mutation: that's easy to do (as pointed out by DrDNA), though I am not sure whether that's what you want to see.

 

My final suggestion depends on what kind of project this is (e.g. practical course, undergrad studies, etc.).

You can, for instance make random mutants on a global scale using marker carrying mutations (e.g. transposons) and then select for mutant strains that become UV resistant. Using the marker you can then identify the gene that has been mutated. However chances are rather low that mutations will generate resistant strains. And it is somewhat work intensive to create a transposon bank and screen it.

[pine_smile]

Dr. DNA,

 

Thanks a lot. It's very helpful.

 

If all you want to do is see if the UVC casues mutation, I'm pretty sure that it would be much easier to expose bacteria to UVC and grow on selective media, for eg, in the presence of an antibiotic (eg, Strep). You would then compare these to control bacteria which are not exposed and calculate the difference in the number of mutant colonies. I recall that we analyzed potential mutagens this way in my undergrad bacteriology lab too many years ago to count....

 

For example:

"This experiment introduces the simple technique of direct selection for the detection and isolation of certain mutants. This technique involves plating bacteria on a selective medium on which only the desired mutants can grow. Specifically, the experiment involves taking a culture of microbes at a high density and plating them on a rich medium containing the antibiotic streptomycin. Streptomycin inhibits protein synthesis by binding to the small subunit of the ribosome and blocking entrance of initiator N-formyl methionine tRNA into the ribosome, thus preventing the start of protein synthesis. A single mutation in the S12 protein of the small subunit of the ribosome prevents streptomycin from binding, thus causing the microbe to become resistant to the antibiotic. By plating a strain onto a medium containing streptomycin, it is possible to fish out the microbes present that have a mutation in the S12 gene. Please appreciate the power of this selective technique. By performing a very simple experiment, it is possible to fish out the 100 or so cells, in a mixture containing billions, that have a change in a specific gene. Also, note how easy it is for a microbe to become resistant to an antibiotic. While the drug is killing 99.9999% of the microbes present, 0.0001% are able to survive, and these microbes are now resistant to the antibiotic. This explains why drug resistance in microbes can occur so rapidly and is a constant problem in medicine."

 

http://www.bact.wisc.edu/microtextbook/index.php?module=Book&func=displayarticle&art_id=127

 

Dear CharonY,

 

Thank you very much for your suggestions. Actually I am designing this experiment (this one of the experiments) for a grant application.

 

I am still not quite clear what the objective of the experiment is supposed to be.

Do you think that radiation might cause mutations that in turn increase resistance to UV radiations? Improbable, but not impossible. However you will only end up (if you are lucky) with UV resistant strains. To demonstrate which mutations were caused is not possible within a normal project budget though (less if it is some kind of course work or undergrad project).

 

Let's go to the suggestions:

- Sequence the genome: in theory it would work, but the price tag and effort to sequence even a small bacterial genome is enormous with traditional Sanger. Newer parallel sequencing methods based e.g. on pyrosequencing are cheaper, but still cost around 15-20k$ per single run. That's about the budget for a phd student for a year.

-use BLAST: BLAST is basically a quick global alignment program used to identify homologous genes/proteins. It is unsuited to detect point mutations (the most likely mutations caused by UV radiation). But regardless, as you got no idea what is going to be mutated you either have to sequence the whole genome, or pick up potential genes involved in UV protection (the P. aeruginosa genome is published), make PCR and check the products for mutations.

- demonstrate that UV causes mutation: that's easy to do (as pointed out by DrDNA), though I am not sure whether that's what you want to see.

 

My final suggestion depends on what kind of project this is (e.g. practical course, undergrad studies, etc.).

You can, for instance make random mutants on a global scale using marker carrying mutations (e.g. transposons) and then select for mutant strains that become UV resistant. Using the marker you can then identify the gene that has been mutated. However chances are rather low that mutations will generate resistant strains. And it is somewhat work intensive to create a transposon bank and screen it.

[Tearfeather]

I am still not quite clear what the objective of the experiment is supposed to be.

Do you think that radiation might cause mutations that in turn increase resistance to UV radiations?

 

that's exactly what pine_smile wants to do, (he explained it to me in private message), like you said ''improbable, but not impossible'', very interesting hypothesis.

I agree this project require a big budget, I don't know if it worth it, especially if you want to do it as you graduate project. In molecular biology work you need a lot of patience, and luck:), it may take time till you will get your first observations, which could tell you if your theory was correct, and time is very

limited and important in graduate work.

But if you need to do it to a company or like undergraduate work, maybe its worth to try:).

[pine_smile]

Dear Dr. DNA,

 

Is it possible that UVC-reisitant bacteria do not resist to antibiotics? If it is case, the UVC mutants may not survive in the selective media with antibiotics anf the mutants may not be selected? Please advise.

 

Thanks a lot.

 

If all you want to do is see if the UVC casues mutation, I'm pretty sure that it would be much easier to expose bacteria to UVC and grow on selective media, for eg, in the presence of an antibiotic (eg, Strep). You would then compare these to control bacteria which are not exposed and calculate the difference in the number of mutant colonies. I recall that we analyzed potential mutagens this way in my undergrad bacteriology lab too many years ago to count....

 

For example:

"This experiment introduces the simple technique of direct selection for the detection and isolation of certain mutants. This technique involves plating bacteria on a selective medium on which only the desired mutants can grow. Specifically, the experiment involves taking a culture of microbes at a high density and plating them on a rich medium containing the antibiotic streptomycin. Streptomycin inhibits protein synthesis by binding to the small subunit of the ribosome and blocking entrance of initiator N-formyl methionine tRNA into the ribosome, thus preventing the start of protein synthesis. A single mutation in the S12 protein of the small subunit of the ribosome prevents streptomycin from binding, thus causing the microbe to become resistant to the antibiotic. By plating a strain onto a medium containing streptomycin, it is possible to fish out the microbes present that have a mutation in the S12 gene. Please appreciate the power of this selective technique. By performing a very simple experiment, it is possible to fish out the 100 or so cells, in a mixture containing billions, that have a change in a specific gene. Also, note how easy it is for a microbe to become resistant to an antibiotic. While the drug is killing 99.9999% of the microbes present, 0.0001% are able to survive, and these microbes are now resistant to the antibiotic. This explains why drug resistance in microbes can occur so rapidly and is a constant problem in medicine."

 

http://www.bact.wisc.edu/microtextbook/index.php?module=Book&func=displayarticle&art_id=127

 

Dear CharonY,

 

I was just told by our hospital (MGH, Boston, MA) that the DNA sequencing only costs $0.40 per run here. We have this service in our hospital.

 

http://www.hpcgg.org/Sequence/submission.jsp?name=servicesforresearchers&subname=sequence

 

 

 

I am still not quite clear what the objective of the experiment is supposed to be.

Do you think that radiation might cause mutations that in turn increase resistance to UV radiations? Improbable, but not impossible. However you will only end up (if you are lucky) with UV resistant strains. To demonstrate which mutations were caused is not possible within a normal project budget though (less if it is some kind of course work or undergrad project).

 

Let's go to the suggestions:

- Sequence the genome: in theory it would work, but the price tag and effort to sequence even a small bacterial genome is enormous with traditional Sanger. Newer parallel sequencing methods based e.g. on pyrosequencing are cheaper, but still cost around 15-20k$ per single run. That's about the budget for a phd student for a year.

-use BLAST: BLAST is basically a quick global alignment program used to identify homologous genes/proteins. It is unsuited to detect point mutations (the most likely mutations caused by UV radiation). But regardless, as you got no idea what is going to be mutated you either have to sequence the whole genome, or pick up potential genes involved in UV protection (the P. aeruginosa genome is published), make PCR and check the products for mutations.

- demonstrate that UV causes mutation: that's easy to do (as pointed out by DrDNA), though I am not sure whether that's what you want to see.

 

My final suggestion depends on what kind of project this is (e.g. practical course, undergrad studies, etc.).

You can, for instance make random mutants on a global scale using marker carrying mutations (e.g. transposons) and then select for mutant strains that become UV resistant. Using the marker you can then identify the gene that has been mutated. However chances are rather low that mutations will generate resistant strains. And it is somewhat work intensive to create a transposon bank and screen it.

[CharonY]

I was just told by our hospital (MGH, Boston, MA) that the DNA sequencing only costs $0.40 per run here. We have this service in our hospital.

 

LOL, sorry, pine_smile, but a single run of conventional sequencers (I was refering to 454 sequencers, which are a complete new breed) will give you only ~500 bp. P. aeruginosa has 6,588,339 bps. Moreover you cannot simply sequence whole chromosomal DNA, but you got to create a DNA library first, subclone it into sequence vectors, get a decent coverage (at least 10x the genome size) assemble the sequences etc.

Whole genome sequencing to detect a single point mutation is not an option, trust me on that. You can only amplify regions of interest and sequence that (don't forget to use proof-reading polymerases). But you need an idea which regions to look at.

Alternatively, you can try a microarray approach (as the genome of P. aeruginosa is available) and just hybridize your mutant strain against it. However you need an array with a decent coverage (most likely you got to design an own format).

[pioneer]

The two base pairs, each only have one lowest energy way to hydrogen bond. If the bases are incorrectly paired, that means the DNA double helix will have points of higher energy potential or hot zones within the double helix. I can see how UV radiation could add energy, and causes hot zones to form easier. When the UV is removed, these can't lower energy, but are stuck there. From there, it depends if the sense strand is still good.

 

Conceptually, a faster way to find these, is to look directly for the hot zones. There will only be four possible signals with two possible good and two possible bad. This could be done at the level of H-bonding energy. Once you locate them, then the search is narrowed for the traditional gene search. One could eliminate sectors up front, allowing large DNA to be reduce to 1-10%.. One may be able to do this with a modification of NMR, where you take a nice chunk of DNA, and see if any of the characteristic peaks are there. This may not tell you where, but could be used to narrow down the amount of DNA to look at. One would have to physically make all the hot spot environments, first, to calibrate the mechanism.

[DrDNA]

Dear Dr. DNA,

 

Is it possible that UVC-reisitant bacteria do not resist to antibiotics? If it is case, the UVC mutants may not survive in the selective media with antibiotics anf the mutants may not be selected? Please advise.

 

Thanks a lot.

 

This is the part where I am unclear. It says in the OP that your hypothesis is that UVC causes mutation.

The the test I gave you will select for mutations...and you can test your hypothesis with that test......but not UVC resistance.

 

If UVC does in fact effieciently kill your wildtype bacteria and you think that it will mutate them, then perhaps expose some to a little UVC, plate, expose these to a little more UVC, plate, expose these a little more UVC, etc etc. Until you obtain some UVC resistant "mutant" strains that survive well in the presence of UVC.

Finally compare how many of the original, unexposed wildtype colonies (negative controls) live after a strong dose UVC exposure to the number of "mutant" colonies that live after the same dose.

 

You should also do some positive controls at the same time. That is expose the bacteria to known mutagens and see how they thrive in the UVC.

Another control could be to expose the wildtype bacteria to UVC and see if they can grow in the presence of antibiotics as described in the experiment I pasted.

 

Of course you will need to figure out what kind of reps that you will for a desired level of statistical confidence, there will probably be significant trial and error, etc.

[pine_smile]

Thanks, Dr. DNA. Your response makes a nice sense.

 

This is the part where I am unclear. It says in the OP that your hypothesis is that UVC causes mutation.

The the test I gave you will select for mutations...and you can test your hypothesis with that test......but not UVC resistance.

 

If UVC does in fact effieciently kill your wildtype bacteria and you think that it will mutate them, then perhaps expose some to a little UVC, plate, expose these to a little more UVC, plate, expose these a little more UVC, etc etc. Until you obtain some UVC resistant "mutant" strains that survive well in the presence of UVC.

Finally compare how many of the original, unexposed wildtype colonies (negative controls) live after a strong dose UVC exposure to the number of "mutant" colonies that live after the same dose.

 

You should also do some positive controls at the same time. That is expose the bacteria to known mutagens and see how they thrive in the UVC.

Another control could be to expose the wildtype bacteria to UVC and see if they can grow in the presence of antibiotics as described in the experiment I pasted.

 

Of course you will need to figure out what kind of reps that you will for a desired level of statistical confidence, there will probably be significant trial and error, etc.

 

Sorry, CharonY. It was me who misunderstood something. people in the hospital clarified this for me as well. Thanks again, CharonY.

 

LOL, sorry, pine_smile, but a single run of conventional sequencers (I was refering to 454 sequencers, which are a complete new breed) will give you only ~500 bp. P. aeruginosa has 6,588,339 bps. Moreover you cannot simply sequence whole chromosomal DNA, but you got to create a DNA library first, subclone it into sequence vectors, get a decent coverage (at least 10x the genome size) assemble the sequences etc.

Whole genome sequencing to detect a single point mutation is not an option, trust me on that. You can only amplify regions of interest and sequence that (don't forget to use proof-reading polymerases). But you need an idea which regions to look at.

Alternatively, you can try a microarray approach (as the genome of P. aeruginosa is available) and just hybridize your mutant strain against it. However you need an array with a decent coverage (most likely you got to design an own format).

[CharonY]

But man, 0.40$ for a genome. That would be awesome. I'd probably sequence everything I come across