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question on pcr product in control DNA
Can someone please help me on this
I am working on this pcr
setup where I have three pcr
primers that will amplify 3
exonic regions of the human
genomic DNA given by a customer.
The customer didn't submit any
control dna, so my boss said to
use any control DNA I have
available. I did so, using a
bacterial dna (S. aureus strain) as my control, and
I have product in the control
dna from each of the 3 primers.
what I don't understand is how
the control dna gave pcr product
when it is a different species
from my customer dna and also
because the pcr primers amplify
specific exonic regions, how did
the control dna also show amplified material?
Re: question on pcr product in control DNA
What kind of control are you planning to do?
A positive control is to indicate that the PCR process is working perfectly with all the components added (excluding the primer and template), so having a PCR product is a good thing. While a negative control (minus a key ingredient) indicates that there is no contamination, which I don't think is the control you're conducting in the experiment above.
Amplification happens when the primers bind to a specific site on the DNA template of the sample. Assuming that you practise good laboratory techniques, there is no contamination/carry over and the PCR is working perfectly, it could be due to:
1. The primers are non-specific and there happens to be a similar site on the bacteria genome which where one or both of the primer pairs could anneal to.
2. The primers are targetted to an important gene which is conserved to some degree in bacteria and humans.
3. Your positive control bacteria genome has been contaminated with DNA of some other organism (not necessarily human).
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|control , dna , pcr , product , question|
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