I have designed genome specific primers for a Na+ transporter gene in wheat. The primers work well on genomic DNA but on cDNA as a template they produce non-specific bands. I have synthesized cDNA using oligoDT primers.
It is acceptable if you have nonspecific bands on genome .your case is strange. is your non specific band very strong? and did the non specific band migrate faster or slower? try to increase a little bit of the annealing time as well as the time for extending. if these do not work, that means the cDNA is not very clean, which may come from incomplete of cDNA synthesis.
Hi
Thank you very much for the quick reply. I expected a fragment of 700bp but what I got was 500 bp. My cDNA looked fine with GAPDH primers. I sequenced the non-specific band anyway and there is no alignment with the gene of my interest. I do not understand what you meant by “ incomplete cDNA synthesis”. Could you kindly explain it?
Thank you
Cheer
chandima
Dear Chandima, You may have designed primers using DNA sequence of the gene of interest. The DNA sequence consists of introns, exons and UTRs. Primers were binding at exon but in between two exons there may be a intron. The total size of amplified product became 700bp. Since cDNA comes from coding sequence (mRNA) only which does not have introns (the introns are removed by post transcriptional modification of mRNA), so the size of the same gene becomes smaller due to lack of intron while using cDNA as template. This may be the reason the product is smaller i.e. 500 bp.
So i would suggest you, is first find out the number of introns in your gene of interest. length of intron, and total length/size of cDNA sequence (coding sequence) as well as DNA sequence of your gene of interest.
if the cDNA has no problem but the 500bp is another gene, I think you have to check the primers. As Patel said the genome and cDNA is not the same. But usually if it works on cDNA, it also should work on genome. The converse is not necessarily true.
- Badges
- Science topic
- Similar topics
- Biological Science
- Bioscience
- Biotechnology