gDNA or cDNA amplification in RT-PCR - (Apr/03/2009 )
I just finished up my bachelor's degree and it's been a while since I dealt with this stuff so please don't mind my basic questions.
I'm getting hung up on something that's probably so simple to understand. When you desing primers for RT-PCR they tell you to design them so that they're intron spanning. They say you do this so only cDNA get's amplified.
This makes no sense to me. When you extract your RNA it's totally possible for a little gDNA to get into the sample. When you convert it to cDNA the gDNA is still there. And when you amplify, the primers will still bind to the exon sequences of both the gDNA and cDNA, therefore ampifiying both right?
I do a DNase treatment step to minimize this but how does designing intron-spanning primers deter ampification of gDNA?
Since the gDNA will still have the introns, while the cDNA does not, designing primers that will span exon-exon junctions ensures that the primers cannot associate with gDNA since the primers will have a sequence that does not exist in the gDNA. Even if they are still able to weakly associate with the gDNA, the amplicon size would be completely different between the cDNA and gDNA products due to the presence of the intron in the gDNA.
Primer 1 designed against exon 6 and exon 7 junction of gene X
cDNA for gene X
gDNA for gene X
Exon1-Intron-Exon2, etc. Exon6-Intron-Exon7-Intron-Exon8
It`s a matter of efficiency of amplification.
If your primers are inside the exons the size of amplification fragment will be smaller when you amplify from cDNA right?. Which means that the amplification of these fragments will be more efficient than the amplification from gDNA. In the firts cycles you do have some amplification of the gDNA (bigger fragment) but after few cycles the smaller fragment will win the competition inside the tube for amplification.
yeah that makes sense.
Dr Teeth, I can understand why it would be beneficial to design a primer at a splice junction but I guess I misinterpreted what was meant by intron spanning. But I understand how a shorter amplicon can be more efficient.