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PCR amplifying non-specific fragment
I have recently designed a set of primers to amplify a fragment of a gene present in an cDNA template. I have performed a blast search on my primers and the results have shown a hit with my gene.
However, after conducing the PCR and cloning my fragment into a vector, i sent it off for sequencing. The results were surprising. Sequence analysis showed that my fragment was from a completely unrelated gene.
I was wondering, bearing in mind my designed primers are specific for my fragment of interest, why i amplified a non-specific fragment.
Any suggestions would help.
Re: PCR amplifying non-specific fragment
There can be two possibilities:
1. There is another gene/DNA fragment with a sequence similar to your primer design.
Geting a hit is not good enough. Is it a conserved sequence which is easily found in other genes as well? What is the E-value?
The DNA primers can't discrimanate between genes, they just bind to the most similar sequences. If that sequence or a small mismatch of it can be found in another gene, your primers might bind to it instead; which leads to:
2. The primers bind to the most abundant/similar regions of DNA. Let me give you an example.
E.g. Your primers is ATCGATCGATCG.
There exists a DNA fragment in your sample, say UAGCUAGCUCGC. There is one mismatch. However, this DNA fragment is present in a larger concentration than your desired fragment. One mismatch is not a big deal and still bind stably at very close temperature to non-mismatch binding.
Say this mismatched DNA fragment is found in 100 copies, while your desired gene is only 10 copies.
After 1 round:
After 3 rounds:
Bear in mind that the amount of primers in your PCR mix is very high, and PCR operates on an exponential basis. The number of mismatched amplificaitons will easily overwhelm the number of specific amplifications.
|amplifying , fragment , nonspecific , pcr|
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