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

Hi, I want to detect a certain bacteria by 16srRNA. I have two questions need your help:

1. how can I find the specific domain of this bacteria, so that I could design primers about this domain? Are there software or websites?

2. I see in papers people use different universal primers. I wonder how should I choose among them?

Thanks a lot! Look forward to your response!

As this sounds like a homework question to me, no straightforward answer

But try to answer the following questions for yourself:

1. think about the tree of life and the variability of gene regions including 16S - does this answer your question about universal primers?

2. which bacteria do you want to detect and how specific do you need to be? i.e. sub-species level/species/genus/family/......

3. what is already known about this specific taxon, i.e what does literature say about your target group (ignoring literature on all not closely related groups)?

4. is your question answered now, did you find primers and or traget genes?

5. if not: try to specify the part which you do not understand and post it here again!

Hi, Thanks for your response! I like the way you answer questions! It makes people learn more.

1. I see the bacteria are devided into 25 main groups based on the 16S rRNA trees. I want to confirm that the 25 main groups have same conserved domain, so the universal primers are same. Am I correct?

2. I want to detect at species level. I have got the DNA sequence for encoding 16sr RNA. I know that 16sr RNA gens have alternating conserved domains and variable domains. I think I need to figure out which part is variable domains, and design primers based on these domains. In this way, I can detect the bacteria specifically. But I don't know how I can identify the variable domains. Are there softwares that I should use?

3.besides, my sample contain human genomic DNA and bacteria DNA. When I design primers, should I avoid the binding of primer to human genomic DNA? But I think it is very hard.
Thanks very much! Look forward to hear from you!

gebirgsziege on Wed Mar 28 08:28:57 2012 said:

As this sounds like a homework question to me, no straightforward answer But try to answer the following questions for yourself: 1. think about the tree of life and the variability of gene regions including 16S - does this answer your question about universal primers? 2. which bacteria do you want to detect and how specific do you need to be? i.e. sub-species level/species/genus/family/...... 3. what is already known about this specific taxon, i.e what does literature say about your target group (ignoring literature on all not closely related groups)? 4. is your question answered now, did you find primers and or traget genes? 5. if not: try to specify the part which you do not understand and post it here again!

Have you checked this: http://depts.washing...ts/bctseq.shtml and general wikipedia page: http://en.wikipedia....S_ribosomal_RNA ?

Also: make sure to check this == > http://bioinfo.unice.fr/biodiv/16S_New_bacterial_species/16S_rRNA_Identification_of_a_bacterial_species.html

1. universal primers are the same, this is the important thing: the primers are the same (conserverd region), but whats "between" the primers is different, and its this difference that makes you able to detect what kind of bacteria it is.

2. Well, you sequence it to know it. And then, when sequenced, you can check it with some databases to see if there is a match with what you got.

3. And yes, you need primers for the bacteria DNA, nbot the human DNA. But just check the wikipedia and other pages.. normally it should be clear to you, how it works. And in the end: what do you want with a PCR? Exaclty what you ask: only amplify that what you want to amplify..so yeah, there are specific primers for specific organisms.. thats just the entire idea of PCR.

Hi, Thanks for your response and very good suggestions. Basically, I want to compare the level of a certain bacteria between two groups of people. I need to design the specific primers for this bacteria, but I don't know how to do it. I don't know how to figure out the variable regions of this bacteria 16sr RNA, as I need to design primers about the variable region. Would you please give me some suggestions?

Thanks a lot!

pito on Wed Mar 28 17:20:27 2012 said:

You dont need to "know" the variable regions, these are the regions you are going to look for (you are going to amplify them with the PCR). WHat you need are the primers for the conserved regions.
You need those primers so you can amplify all the bacteria present and then you can (based on the variable regions) check what types of bacteria are present.
Unless of course you know, up front (before you start sequencing/pcr), what bacteria you look for ==> then you can use specific primers based on the DNA of the bacteria you are looking for.

For example: if you are only intersted in knowing whether bacteria 1 and 2 are present, you can look for specific primers for those 2 bacteria.
You can find them in the literature or databases.
I dont really think you will need to desing your own primers.. there are allready a lot of primers out there.
If you do need to desing primers then you need to look up some information on "how to design primers" (but this is something different really, designing primers is a subject on itself and there are helpfull websites for this, but to start doing this: you need to know the DNA sequence of the bacteria you are looking for!)

Anyway, ncbi database can help you.
And check the followig links:

http://www.plosone.org/article/info:doi%2F10.1371%2Fjournal.pone.0007401
http://www.bioinformatics-toolkit.org/index2.html
http://www.wjgnet.com/1007-9327/pdf/v16/i33/4135.pdf ==> this is essentially what you want...
http://cmliid.org/pdfs/UniversalPCRFactSheet.pdf
http://www.lutzonilab.net/primers/page604.shtml
http://mybio.wikia.com/wiki/Primer_design_tools ==> a lot of good links to start looking for more information.




And a short answer to your question: the variable region (or DNA sequence in general) can be found using a database like NCBI. Just type in 16S RNA and see what you get.. (in the search menu)

joy123 on Wed Mar 28 17:52:15 2012 said:

Thanks for your response! Yes, I need to design primers for the specific bacteria, and as it is seldomly studied, I don't see any report about its primers published. I will read the links you showed me. And please let me know if you have further suggestions! I appreciate your time on it!

pito on Wed Mar 28 18:42:47 2012 said:

Well, I am a bit confused because either its about 16S RNA sequencing (identifying bacteria) or its about identifying a known bacteria... You first started with the 16S RNA story and now you changed to identifying a bacteria that is known...

You know the bacteria, right? Do you know the sequence?
If you know the sequence, then based on that sequence you can design primers using the links I gave you.

joy123 on Thu Mar 29 15:10:23 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!

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