Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo

how to design primers for 16sr RNA


  • Please log in to reply
24 replies to this topic

#1 joy123

joy123

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 105 posts
3
Neutral

Posted 27 March 2012 - 09:47 AM

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!

#2 gebirgsziege

gebirgsziege

    I like fungi

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 392 posts
25
Excellent

Posted 28 March 2012 - 12:28 AM

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!
A man cannot be too careful in the choice of his enemies. (Oscar Wilde)

#3 joy123

joy123

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 105 posts
3
Neutral

Posted 28 March 2012 - 07:43 AM

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!Posted Image


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!



#4 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,328 posts
80
Excellent

Posted 28 March 2012 - 09:20 AM

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...al_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.

Edited by pito, 28 March 2012 - 09:26 AM.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#5 joy123

joy123

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 105 posts
3
Neutral

Posted 28 March 2012 - 09:52 AM

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!

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...al_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.



#6 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,328 posts
80
Excellent

Posted 28 March 2012 - 10:42 AM

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.o...al.pone.0007401
http://www.bioinform...org/index2.html
http://www.wjgnet.co...16/i33/4135.pdf ==> this is essentially what you want...
http://cmliid.org/pd...CRFactSheet.pdf
http://www.lutzonila...s/page604.shtml
http://mybio.wikia.c...er_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)


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!


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...al_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.


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#7 joy123

joy123

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 105 posts
3
Neutral

Posted 29 March 2012 - 07:10 AM

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!


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.o...al.pone.0007401
http://www.bioinform...org/index2.html
http://www.wjgnet.co...16/i33/4135.pdf ==> this is essentially what you want...
http://cmliid.org/pd...CRFactSheet.pdf
http://www.lutzonila...s/page604.shtml
http://mybio.wikia.c...er_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)



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!


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...al_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.



#8 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,328 posts
80
Excellent

Posted 29 March 2012 - 10:24 AM

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.


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!


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#9 joy123

joy123

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 105 posts
3
Neutral

Posted 29 March 2012 - 10:45 AM

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!

Edited by joy123, 29 March 2012 - 10:46 AM.


#10 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,328 posts
80
Excellent

Posted 29 March 2012 - 11:06 AM

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?

Edited by pito, 29 March 2012 - 11:06 AM.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#11 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,442 posts
243
Excellent

Posted 29 March 2012 - 11:22 AM

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!

#12 joy123

joy123

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 105 posts
3
Neutral

Posted 29 March 2012 - 11:35 AM

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!


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!



#13 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,442 posts
243
Excellent

Posted 29 March 2012 - 11:55 AM

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.

#14 joy123

joy123

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 105 posts
3
Neutral

Posted 29 March 2012 - 12:03 PM

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?


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?



#15 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,328 posts
80
Excellent

Posted 29 March 2012 - 12:11 PM

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.

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?



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?


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.





Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.