I designed one primer pair in exon-exon junction. My forward primer is in exon, but the reverse primer is in exon-exon junction. My primer designer is NCBI-PrimerBLAST.
My PCR product was run in Agarose gel. Positive cDNA control is LnCap cDNA, and gDNA control is gDNA from blood.
I think one possibility is that you may have pseudogenes to your gene of interest. Might just have to design some new primers (I'd recommend aiming for a different area on the gene). Good luck!
For PCR amplification the nucleotide base composition and their complementarity in primers at its 3' end is very important. In case of genomic DNA may be the primer designed at exon-exon is amplifying only the region of same size of amplicon which is expected in cDNA due to sequence of junction primer at its 3' end perfectly complimentary to template sequence at the end to first exon rest of sequence in primer is acting as tail. This may be the possible reason for amplification of same size of amplicon in both genomic DNA and in cDNA. The size of intron between two exon also matters for amplification.
Thank you for your answers and advices.
My PCR product is 202 bp.
I think, I find the problem, because I designed reverse primer (20 bp) in exon 7- exon 8 junction, but only 2 bp there is in exon 7, and 18 bp in exon 8. And 1 bp sequence of 2 bp is identical in intron.
If I amplify the gDNA, my PCR product should be ~480 bp.
But I can't understand, why is the same PCR product size?
But I am designing primer pair again!
Hello,
I think the pseudogenes are the main problem. Blast your desired gene sequence against the human genome and transcriptome on NCBI and then look if pseudogenes are found (ctrl+F type in pseudo is the fastest method ;-) ).
In the case there are one or more found copy the sequence and use clustalw2 to identify complementary areas of your gene of interest with its pseudogene/s.
Normaly you should find an area where no overlap is present and this sequence you can use to design your primer. If there is a nearly complete match it is enough when you place the last 5 (better 8) bp of your froward or reverse primer where they have a mismatch with the pseudogene.
5-8 bp mismatch at the 3´ end of an primer should reduce the amplification around 99%, this should be enough to avoid amplification of the pseudogene.
Even when you design exon-exon spanning primers 5-8 bp of the 3´ end should be situated in the exon where the extension continues.
When you have only the first 2 bp of the 5´ end in the first exon the polymerase is able to extend this and fills up the missing 2 bp after several cycles.
When you have 5-8 bp in the second exon and the primer missanneals the unmatched 5-8 bp function like a ski jump when the polymerase comes along and drop it of and avoid amplification of unspecific amplicons.
I hope this is helpful :-)
The Blat utility at the UCSC genome browser:
http://genome.ucsc.edu/cgi-bin/hgBlat?hgsid=436128113_P9IhDmmDYzleRPN9tZTbq3deLH46&command=start
is a very fast way to determine if you have a processed pseudogene that is causing your problem. Paste the predicted sequence of your PCR product into the Blat window and choose the organism. Hit submit and you will get a list of genomic locations with matches to your sequence. You can look at each of these to determine if your primers are likely to amplify that genomic sequence.
thank you guys:) My problem was solved:) The pseudogene is a guilty :)
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