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Ghost gene


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#1 mjones

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Posted 26 October 2010 - 12:35 PM

Hello,

Maybe someone can help me with this situation.

I have a gene (approximately 600bp) that is positive by PCR and sequencing has confirmed that our target gene is present. The problem is that when we repeat the pcr of the same samples the gene "disappeared" and we don't have any idea how that is possible. It has appeared in different dna extractions, different pcr and even with different primers so it cannot be a contamination. My problem is that results are being very difficult to reproduce, it seems like a matter of chance instead of a scientific result.

Anyone had already experienced a situation like this?
Thank you in advance.

M. Jones.

#2 bob1

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Posted 26 October 2010 - 02:31 PM

It sounds like you have a contaminating inhibitor in the PCR that is blocking the reaction sometimes (perhaps due to minute changes in the dilution of the DNA). Try doing a "DNA gradient" where you do serial dilution of your stock DNA and see if diluting helps the signal.

Another option is that the PCR is at its limits i.e. the sequence is only just being detected and it is a bit luck of the draw as to whether you see it from one reaction to the next.

#3 mjones

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Posted 27 October 2010 - 02:01 AM

It sounds like you have a contaminating inhibitor in the PCR that is blocking the reaction sometimes (perhaps due to minute changes in the dilution of the DNA). Try doing a "DNA gradient" where you do serial dilution of your stock DNA and see if diluting helps the signal.

Another option is that the PCR is at its limits i.e. the sequence is only just being detected and it is a bit luck of the draw as to whether you see it from one reaction to the next.



Hi,

Thank you for your suggestions. I also think that some inhibitor is blocking the reaction... and we already tried MgCl2 gradients, temperature gradients, hot starts, different DNA concentrations, distinct DNA extractions techniques and nothing seems to work... And it happens in all "positive" samples, it's not just one sample with this problem.

But I didn't understood what do you meant with your second option... Can you explain it a bit better?

Thanks again.

M.Jones

#4 bob1

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Posted 27 October 2010 - 03:05 PM

Basically what I was talking about is that for a PCR to work it needs a certain number of copies of the target sequence to start it off. In theory this number is 1 copy, but in practice, depending on the reaction conditions(polymerase used, presence of inhibitors, efficiency of primers etc.) it may be 10 copies or 50 or even a hundred copies. It could be that your reactions are right on the limits of detection for your target, so sometimes you don't see a product, but sometimes you do with the slight variations in the mix as you make it up.




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