pcr amplification - right amplification,right sequence but still in dilemma (Jan/03/2009 )
Hi one and all
I have amplified a pCR product from a cereal plant using the primer pair designed from an oilseed plant, and got the expected amplicon size. cloned it, sequenced and got the correct sequence and now I have a couple of questions to be clarified.
Q1.The primers were designed using cDNA sequence from an oilseed plant but I could amplify the pcr product from cereal plant using genomic DNA as template. how was it possible?????
Q2. I got the same amplicon size even with RT-PCR. does it mean there are no introns in the primers spanning region of the cereal plant????
Q3. The primer spanning region has introns in the oilseed plant. in that case, my amplicon size obtained using genomic DNA as template should give longer amplicon than expected . am I right??
any suggestions are appreciated to justify my results.
In need of an urgent respose.
thanks a million tons
Going by what you say, it seems that there are no introns or the intron identified is actually an exon.
or there is contamination in the PCR. The oilseed plant genomic DNA sample got contaminated with some cDNA from the cereal plant.
Even if its true, the genomic DNA is not zero. So do you see a band running at the size with the intron?
You can do a PCR directed towards another region in the genomic DNA to make sure that the genomic DNA is there.
Or look at the protein somehow, if you can. The sequence and the size of the protein in native and recombinant form will tell you if that intron is there.
But how is it possible that I got the same amplicon size even when i did RT-PCR. Iam sure that the sample is not contaminated as I have tried out with four different sets of DNA samples.