Hi all,
I have to design primers for qRT-PCR on a bacterial gene. I am aware that one should pay attention to exons/introns..but since I am dealing with bacteria, do I need to care about this aspect?How can I check if my gene has exons or introns?
Also, I tried to run Primer Blast by using my gene as an input (the accession number is NC_021078.1) and I selected "primer must span an exon-exon junction". When I try to run the program it returns with
" No primers were found...see explanation below: Exon/exon junction cannot be found for submitted PCR template. Try search again without exon/intron requirements".
Could it mean that my sequence does not contain introns?
Thanks.
Unlike eukaryotes, genes of prokaryotes do not have introns. The entire gene is translated, no introns, no alternative splices etc..Hope this helps
hi
This accession number is NC_021078.1 related to complete sequence of (Klebsiella pneumoniae strain Kp002 plasmid pJEG011) no for 1 gene !?
good luck
As Achraf El Allali said, the prokaryotes genes don't have any introns. You can use the Primer3 software to design your primers.
I agree genes of prokaryotes do not have introns like eukariote.
As in bacteria you do not have introns which could differentiate genomic sequence form mRNA sequence you have to take extra care to avoid genomic DNA contamination of your RNA sample. If you do not get rid of the DNA, you will get amplification both from gDNA and mRNA (cDNA).
To check if your RNA isolate is freee from gDNA always use No RT Control - a sample of your RNA which did not undergo RT. The amplification in this sample means your RNA is contaminated with gDNA and that will have an adverse effect on your qPCR results.
As all answers above explained, no need to worry about introns and exons in bacteria. For primer design I used Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/primer3/) and if I recall correctly I ended up using oligos suggested by the software.
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