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Primer Specificity: Testing only one primer


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#1 djvan

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Posted 20 February 2014 - 01:38 PM

Hi Everyone!

 

I've designed a PCR reaction to amplify a region of the dog genome.  One primer binds to a repetitive element in the dog genome.  So, in order to gain specificity, it is imperative that my other primer be extremely specific to my region of interest.

 

Primer design tools, such as NCBI's, create primer pairs.  The only way I've been able to test the specificity of my primer is to do an nBLAST.  I always get a ton of his to regions that bind my primer non-specificaly - not good.

 

So I ask, is there a program that I can use to design a single, specific primer?  When I use NCBI's tool, I utilize the most stringent parameters.  However, when I then run the primer through nBLAST, it comes up with several hits to other locations....

 

Thanks!



#2 memari

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Posted 21 February 2014 - 09:24 AM

Hi Everyone!

  The only way I've been able to test the specificity of my primer is to do an nBLAST. 

Thanks!

 

Who told you this?

For primers you should use Primer-Blast website.

 

http://www.ncbi.nlm....s/primer-blast/

 

Ccopy and paste your forward and reverse primers inside "Primer Parameters" section

and then in the "Primer Pair Specificity Checking Parameters" section,

select one of these:

1-Refseq mRNA  for Only mRNA

2-Genome

3- Refseq RNA  for all RNAs

 

and you can also select animal of your interest in "Organism" section.

 

Then click on "Get Primers".

nBLAST mislead you 100%.


Edited by memari, 21 February 2014 - 09:30 AM.

-----
Babak Memari

#3 djvan

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Posted 21 February 2014 - 12:01 PM

 

Hi Everyone!

  The only way I've been able to test the specificity of my primer is to do an nBLAST. 

Thanks!

 

Who told you this?

For primers you should use Primer-Blast website.

 

http://www.ncbi.nlm....s/primer-blast/

 

Ccopy and paste your forward and reverse primers inside "Primer Parameters" section

and then in the "Primer Pair Specificity Checking Parameters" section,

select one of these:

1-Refseq mRNA  for Only mRNA

2-Genome

3- Refseq RNA  for all RNAs

 

and you can also select animal of your interest in "Organism" section.

 

Then click on "Get Primers".

nBLAST mislead you 100%.

 

Hi Memari-

 

I'm trying to amplify a region of unknown sequence (a new animal model).  I know the DNA sequence on one end of the deletion.  I'm using the known sequence to design one of my primers.  The other primer is designed to bind to a repetitive element in the dog genome.  Running primer design won't work, because only the WT dog genome is available to check it against.

 

I'm hoping to amplify the unknown area, and send it for sequencing.  My reverse primer is not specific - it's created that way.  Therefore, it's imperative that my forward primer is highly specific.  Is there a way to test my forward primer specifcity outside of nBLAST?  Or rather, a tool I can use to design the a primer to the known sequence area that is highly specific (in the dog genome).

 

Thanks!



#4 Ameya P

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Posted 22 February 2014 - 04:47 AM

Djvan, 

 

Since Primer Blast lets you enter primer sequences of your choice, you could simply enter the primers you have designed and check the variability of PCR products generated.  

 

You would want to sequence the PCR product generated to know the sequence of the region, right. So, since sequencing read wont be longer than 800 bp, you could use Primer Blast to confirm that PCR products generated with your primer pair are in the neighbourhood of 800 bp. 

 

I am not a big fan of Primer Blast myself, so I prefer the In-silico PCR at UCSC. Here you can select the Organism of your choice, enter your primers and see, if the PCR product is similar to what you are looking for. Else, design a new primer from the sequence you know so far. 

 

If what ever I have said so far, is of no use to you, please provide more preliminary information of what you are aiming to do, so that the 'Elders' here can help you out.  :) 


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#5 phage434

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Posted 22 February 2014 - 10:01 AM

I would not worry too much about other matches to a genome, unless those matches were also close to (and in the correct orientation wrt) my other primer. Your best bet is to try this, and see what happens. The match at the 3' end of the primer matters most. Aim for relatively high GC (60-70%). Avoid hairpin structures which bind the 3' end and leave a 5' extension. Use the IDT tools to identify homo and heterodimers.






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