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how to design primers for 16sr RNA - (Mar/27/2012 )

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Also, why are you focused on the 16s region of your genome. There are other regions of the genome which are much more specific for the bacteria you are trying to detect. You seem to be insisting on looking at 16s, which is a region carefully chosen to be as similar as possible among all bacterial species, yet you are trying to find something unique in it.
Look elsewhere!

-phage434-

Thanks for your response! Would you please give me some suggestions how to find the "other regions of the genome which are much more specific for the bacteria you are trying to detect"? My bacteria haven't been well studied, but the genome is know. Is there software to search the specific regions for detecting this bacteria? Thanks!


phage434 on Thu Mar 29 19:22:27 2012 said:


Also, why are you focused on the 16s region of your genome. There are other regions of the genome which are much more specific for the bacteria you are trying to detect. You seem to be insisting on looking at 16s, which is a region carefully chosen to be as similar as possible among all bacterial species, yet you are trying to find something unique in it.
Look elsewhere!

-joy123-

Well, if you have sequence, then you can choose a gene that occurs rarely in other species. Look for "hypothetical protein" and blast the protein sequence against the protein database (or you can use "Blink" which has pre-computed this result). If the only hits are in your target organism, you have a unique gene which you design primers for. For best results, I'd do this with several genes and do PCR with all of the primer pairs, as a cross-check.

-phage434-

I haven't checked "hypervariable regions of the 16sr RNA" in my bacteria, as I don't know how to do it.

Do you mean, I should align the universal primers (forward and reverse) to my bacteria 16sr RNA sequence, to find the conserved regions. And the sequences between the conserved regions are the hypervariable regions. Then, I could design primers using any two of the hypervariable regions. And do PCR, run gel, to see if the product show the length between the two regions/primers. Am I correct?

pito on Thu Mar 29 19:06:39 2012 said:


joy123 on Thu Mar 29 18:45:45 2012 said:


Sorry to confuse you. Yes, I want to test the level of a specific bacteria that I know the 16srRNA sequence. I have problem with designing the bacteria specific primers so I posted this poster to get some help.

According to my understanding, I need to find the variable regions of the 16srRNA sequence, and then design primers based on these variable regions. And then, I should check the primer specificity on "ribosomal database project" -->"probe match". Am I correct?

But I don't know how to find the variable regions. Sorry I have checked the links you showed me, but still not so clear how to find the regions (I tried some softwares but they don't work). Are there free online tools to do it? Would you please give me more clue?

Thanks a lot!

You do know the sequence right?
Have you compared this sequence with the general primers allready?

Take for example:

http://www.ncbi.nlm....73153&to=474707 (16S RNA seq of Staphylococcus aureus RF122) And now use the B27F primer, see if you can find this primer in that sequence...
you should be able to find it...
now think about it: its a general primer for the conserver region.. then where is the "non conservered" region...

Do the same excercice with your bacteria!
Find the 16S rna, see where the random primers bind and then think wether you can make a primer for not conservative region...


Have you done a search on "hypervariable regions of the 16S RNA" of your bacterium?

-joy123-

Google it, you will find what hypervariable regions of the 16 S RNA means..
And yes, what you propose, is indeed an option.. you could indeed design the primers like that. ANd for example: check if the primers (the ones you make) align with other bacteria (using those databases and blast them.., its like phage434 also suggests: blast a possible specific gene and see if you get a lot of hits... if not: you know its pretty unique.

joy123 on Thu Mar 29 20:03:08 2012 said:


I haven't checked "hypervariable regions of the 16sr RNA" in my bacteria, as I don't know how to do it.

Do you mean, I should align the universal primers (forward and reverse) to my bacteria 16sr RNA sequence, to find the conserved regions. And the sequences between the conserved regions are the hypervariable regions. Then, I could design primers using any two of the hypervariable regions. And do PCR, run gel, to see if the product show the length between the two regions/primers. Am I correct?

pito on Thu Mar 29 19:06:39 2012 said:


joy123 on Thu Mar 29 18:45:45 2012 said:


Sorry to confuse you. Yes, I want to test the level of a specific bacteria that I know the 16srRNA sequence. I have problem with designing the bacteria specific primers so I posted this poster to get some help.

According to my understanding, I need to find the variable regions of the 16srRNA sequence, and then design primers based on these variable regions. And then, I should check the primer specificity on "ribosomal database project" -->"probe match". Am I correct?

But I don't know how to find the variable regions. Sorry I have checked the links you showed me, but still not so clear how to find the regions (I tried some softwares but they don't work). Are there free online tools to do it? Would you please give me more clue?

Thanks a lot!

You do know the sequence right?
Have you compared this sequence with the general primers allready?

Take for example:

http://www.ncbi.nlm....73153&to=474707 (16S RNA seq of Staphylococcus aureus RF122) And now use the B27F primer, see if you can find this primer in that sequence...
you should be able to find it...
now think about it: its a general primer for the conserver region.. then where is the "non conservered" region...

Do the same excercice with your bacteria!
Find the 16S rna, see where the random primers bind and then think wether you can make a primer for not conservative region...


Have you done a search on "hypervariable regions of the 16S RNA" of your bacterium?

-pito-

Dear Joy,

I have performed the PCR of 16sRNA using universal primers. As I know, there are few primer set and you can try them. Since you know the sequence of your bacteriam, you can use restriction enzyme to further identify your bacterium.

-newborn-

I agree with phage454s advice - if you know the sequence, try to look for unique regions in your bacteria, makes live a lot easier.

I do not know your experiment and setup so here is how I would approach this problem:

1. Make the aim of your experiments clear to yourself, make yourself familiar with the methods needed to achieve your target. This might involve a considerable amount of reading scientific papers, so allow yourself enough time for this. Given your questions you should start with basic papers on PCR detection and primer design.

2. try to set up an experimental plan including timing etc. always allow at least 30% more time than you think you will need. Something will go wrong and you will need the extra time, if not, you have some extra time - which is never a bad thing

3. before starting to design primers decide what is more important for your experiment: primer specificity or primer sensitivity! This can be a tricky one when working with samples contaminated with excess amounts of host DNA. 16s has the advantage that it is multi copy and relatively easy to amplify - vs a probably tricky unique region (e.g. high GC or AT content....). Once you have your primers talk to your supervisor or any senior lab staff experienced with these sort of things for what they think about your strategy.

4. If not sure about anything substantial at this point, approach your supervisor and ask for help! Most supervisors prefer to answer questions instead of getting crap results because people do not ask when they are stuck and do what comes to their mind - which results in you having a bad time, as your supervisor spent a lot of money for results that are basically useless! And with all the preparation done in the previous points you will be able to ask informed questions and show that you have worked on the problem. Any decent supervisor will acknowledge this and happily help you.

-gebirgsziege-

Hi! Thanks very much for your advice. I am now searching hypothetical proteins on the PATRIC website. I got lots of hypothetical proteins, and I think I should next blast them in NCBI one by one, and choose the one that is uniquely expressed in my bacteria. Am I on the correct way?

I see you mentioned about "Blink". I think it may save time. I wonder which website have this function? And do you have further suggestions?

Thanks a lot!


phage434 on Thu Mar 29 19:55:06 2012 said:


Well, if you have sequence, then you can choose a gene that occurs rarely in other species. Look for "hypothetical protein" and blast the protein sequence against the protein database (or you can use "Blink" which has pre-computed this result). If the only hits are in your target organism, you have a unique gene which you design primers for. For best results, I'd do this with several genes and do PCR with all of the primer pairs, as a cross-check.

-joy123-

This sounds good. You can find blink on the right hand side of an NCBI protein page, the first link under "Related Information". It will bring up a pre-computed blast of that protein against all other proteins in Genbank, with hot links to alignments. It will also show conserved domains of the proteins.

-phage434-

Thanks!
I searched in NCBI (protein): "XXX(my interested bacteria) hypothetical protein", and got ~1000 results. Among them, about 100 are "conserved hypothetical proteins". Then, I open these hypothetical proteins one by one, and click on the "Blink". I can see how this protein is expressed in "Archaea, bacteria, matazoa, etc." And I need to find the one only expressed in my bacteria. Am I on the right track?

phage434 on Mon Apr 2 19:47:57 2012 said:


This sounds good. You can find blink on the right hand side of an NCBI protein page, the first link under "Related Information". It will bring up a pre-computed blast of that protein against all other proteins in Genbank, with hot links to alignments. It will also show conserved domains of the proteins.

-joy123-
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