unknown sequence and the extra PCR band
I have done my PCR, the expected PCR product size is 772bp (right size,right gene), and I also did cloning and sequence as well. What happened is ?I have extra band (172bp), I blast the sequence on NCBI but I cannot find any similarity. however, from the sequence, I found the forward and reverse primer switch the location, which means my forward primer flip over and located in the end of the sequnce and the reverse one jump to the bigining of the sequence. I also blast the midlle section of the sequcece. I could not find it in the receptor and any other genes.
Can anyone give some commment for the situation?
Re: unknown sequence and the extra PCR band
I understand you already know the sequence of 172bp DNA fragment.
It could be any DNA fragment, even non-coding region etc. (in eucaryotic organisms).
GenBank doesn't have all the possible DNA sequences in database - remember DNA of many organisms haven't been sequenced yet.
172bp is comparatively short sequence. If you assume it's a coding sequence try to amplify larger DNA fragment based on this sequence.
It would be useful to know whether you used as your PCR template the whole genomic DNA, or mitochondrial, plasmid (if bacterial) or just performed nested PCR on isolated DNA sequence. You could try to map your sequence on the genome if you suspect it's worth it.
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