24 replies to this topic

[newborn]

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.

[gebirgsziege]

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.

Edited by gebirgsziege, 30 March 2012 - 12:21 AM.

[joy123]

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 29 March 2012 - 11:55 AM, 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.

Edited by joy123, 02 April 2012 - 11:27 AM.

[phage434]

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]

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 02 April 2012 - 11:47 AM, 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.

[phage434]

Conserved hypothetical means that the protein is found in many other species.  You may want to look for hypothetical proteins that are not conserved.

[joy123]

Dear All, I want to say "thank you" to everybody who helped me in this problem. I am working on it now. I will let you know when I get process! I appreciate your time and kind help!

[joy123]

Hi! I have found some hypothetical proteins that uniquely expressed in my interested bacteria. I designed primers based on a protein sequence, and the PCR result is good.

I have question here: What if the expression level of this protein in the bacteria is different/unstable. In that case, I cannot use it for quantification (qPCR) of the bacteria? As the proteins are hypothetical, there is no study about it and it is hard to say if the protein expression is stable.

Thanks a lot!

phage434, on 29 March 2012 - 11:55 AM, 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.

[phage434]

You are looking at genomic DNA, so the expression level is completely irrelevant.  If the gene is there, your PCR will pick it up.  There will always be one copy per genome (well, perhaps a few during rapid growth).

[joy123]

Do you recommend I try several genes? Thanks a lot!

phage434, on 11 June 2012 - 01:44 PM, said:

You are looking at genomic DNA, so the expression level is completely irrelevant. If the gene is there, your PCR will pick it up. There will always be one copy per genome (well, perhaps a few during rapid growth).