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Unspecific Primer Amplification

Unspecific Primer Amplification - PCR - Polymerase Chain Reaction Forum

Unspecific Primer Amplification - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #1  
Old 08-11-2009, 07:18 AM
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Default Unspecific Primer Amplification



Hello,

I am running 2 multiplex reactions of 5 different prey species that are of genus level relatedness. I have developed species specific primers for 4/5 species, but the 5th species primers always amplify the predator DNA at the exact same size of the expected prey product size. I have tried at least 6 different primer sets and every single one creates a product for the predator that is equal to the prey.

There is no way the predators could have eaten any of this prey species and I have extracted the DNA of multiple predators to be sure there wasn't any contamination. I am pretty new at PCR and don't understand how a very unrelated species could be amplified when other extremely closely related prey species don't amplify the predator DNA.

I guess my main question is why the primers would amplify the predator DNA to a product identical to the desired prey species every time.

I have been trying to troubleshoot this for over a month now and have run out of ideas besides sequencing the predator DNA but the situation just confounds me.
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  #2  
Old 08-12-2009, 09:57 AM
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Default Re: Unspecific Primer Amplification

PCR is a deceptively simple and tricky process. I think your problem boils down to the part where the primer anneals to the template. I've had a similar problem once where I obtained a band whose size is identical to my desired product, but turns out to be a different thing entirely.

What happened was that my 25 bp primer (designed based on a conserved region of my desired product) bound exactly to another a DNA strand of unknown length that happened to have that exact 25 bp sequence complementary to my primer. Highly unlikely, but it happened. So for my sequencing result, I can see the first 25 bp of the unknown product is exactly the same as my primer, but the other 400 bp is completely different. And in PCR, the amount of starting template is crucial, which is why I believed that this unknown DNA strand was in higher abundance compared to my desired product. After a few rounds of PCR, the amount of undesired PCR product generated (ie. templates for subsequent rounds) overwhelms the amount of DNA template that I desired to anneal to my primers.

I'm not sure, but I guess your problem could be similar to mine. Perhaps your predator species happened to have a tiny region of ~20 bp that is highly complementary to your primer (probably in the intron segment/untranslated region of mRNA). My suggestion would be to move on and design a new primer, either in a different conserved region or shifting the position slightly (eg. use move it bout 10 bp left/right of the DNA template).

Hope this helps to clarify your problem a bit.
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  #3  
Old 08-12-2009, 02:25 PM
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Default Re: Unspecific Primer Amplification

It sometimes happens that two different DNA sequences after your PCR have exactly the same or similar length - electrophoresis will tell you only that.
Probably in this case your primers are not specific enough.

It is difficult to design the right pair of primers if the genomes of the organisms you study haven't been fully sequenced yet. You probably checked GenBank database anyway.

The only solution I can think of is to find more conserved sequence and design new primers.
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Old 08-14-2009, 07:11 AM
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Default Re: Unspecific Primer Amplification

Thanks for this site very helpful.
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Old 08-20-2009, 04:09 PM
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Default Re: Unspecific Primer Amplification

Quote:
Originally Posted by nebulus View Post
Hello,

I am running 2 multiplex reactions of 5 different prey species that are of genus level relatedness. I have developed species specific primers for 4/5 species, but the 5th species primers always amplify the predator DNA at the exact same size of the expected prey product size. I have tried at least 6 different primer sets and every single one creates a product for the predator that is equal to the prey.

There is no way the predators could have eaten any of this prey species and I have extracted the DNA of multiple predators to be sure there wasn't any contamination. I am pretty new at PCR and don't understand how a very unrelated species could be amplified when other extremely closely related prey species don't amplify the predator DNA.

I guess my main question is why the primers would amplify the predator DNA to a product identical to the desired prey species every time.

I have been trying to troubleshoot this for over a month now and have run out of ideas besides sequencing the predator DNA but the situation just confounds me.
any new update? seem very interesting... will think when i am dream and working tomolo
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Old 08-21-2009, 09:33 PM
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Default Re: Unspecific Primer Amplification

Well I solved the issue. Even though the other prey species didn't amplify the predator with 5-6/20 bp difference the prey specie's primer pair was still able to amplify it.

I screened the primers again using genbank for at least 8 other members of the predator's genus and made sure there was about 50% difference between the primer's sequence and the predator. Still don't understand why my criteria allowed the other prey species primers to pass the test but not that one.

I am in the process of testing my newly developed prey primers against the other prey species and if they pass that I will finally be done with my multiplex optimizations after 7 months.

Next I need to test for primer sensitivity in the presence of predator DNA via serial dilutions of 1:1 prey solutions in a constant concentration of predator DNA. I will be starting my feeding trials the first weekend of September and looking at detection halflives.

Should be fun, but over 500 PCR reactions not including field trials.
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  #7  
Old 08-23-2009, 12:33 PM
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Default Re: Unspecific Primer Amplification

great to hear that nebulus!!! all the best and if anything, u know where to tell us happy or not so happy news
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Old 08-24-2009, 10:21 AM
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Default Re: Unspecific Primer Amplification

Nebulus, great to hear that you solved the problem. Seem that you're going to have lots of things to do (as we all have ) so good luck

Last edited by Aga; 08-24-2009 at 10:25 AM.
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