Sometimes primers have more than one target sites on the total RNA. This should not be a problem in PCR, as the possibility of both primers fitting to additional DNA fragment is relatively low, especially once you adjust cycling conditions.
However, if your primers are too short or not specific enough, you'll get all sorts of funny stuff.
Usely one primer is not sufficient to be specific for one gene. In a PCR reaction always two primers are used, and even here the PCR product is not always specific for one gene.
So if cdna is synthesised with gene specic primer. It should be impossible to score for genes on some other chromosome right. But when I try I get expected band size of amplicons when I use primers for gene present on a different chromosome. When I blasted the primer sequence and checked they dont seem to have any hits on the chromosome for which am using gene specific primer.
How do I solve this issue. Though am getting desired band could it be that some other gene is getting amplified and not my desired gene. In that case how can the band size be perfect. I think eluting and sequencing the band could be an option. But is there anything else I could do
Could it be, that the primers are partially specific to the off-target sites?
I think you could adjust cDNA synthesis step by changing temperature / time or whatever is suggested by your revertase provider.
Also, it might be that the RNA you're working with is not the same that's being used for blasting. Or that the gene in question is not transcribed under the current growth conditions.
Basically, mutations happen.
The reverse transcriptase reaction is done by 42 or 50 degrees centrigade depending on the kit you use. The annealing temperature of your gene specific reverse primer will be higher around 60 degrees centrigade I suppose. So it is espected that your primer will anneal to multiple targets, also on different chromosomes.
- Badges
- Science method