Hi All,
I am design primers and Probe for qPCR to quantify a gene of bacteria that I will inject into mice. How I determine the target region in my bacteria gene that is not similar with mouse genomic or transcipt to design primers and probes.
I used Prime3plus, PrimerQuest tool to design primer and probe. However, When I check primers by Nucleotide Blast I find that these primers and probe amplify both my target gene and mouse's genomic and transcripts.
it is common to identified bacteria using 16srDNA gene. The gene never exist in eukaryotic organism including mice. Therefore general primer amplifiying 16srDNA fragmen would satisfied. Eukaryotic rDNA will be 18srDNA which should be different to 1srDNA sequence from bacteria. However yu should check whether general primer for amplifiying bacterial 16srDNA will not pick up fragment from 18srDNA. Good luck
Does your gene of interest have multiple exons/introns? It might help to design primers that flank intron regions (e.g. forward primer at the end of exon 1, reverse primer at the beginning of exon 2). That also excludes amplification of genomic contaminations.
We are working on developing PCR based markers for detecting specific isolates and races of Xanthomonas campestris pv. campestris. In our case we have taken the sequences of the bacteria available in the public data bases and align them using MAUVE tool and design the primers on conserved region for races and variable regions for isolates and found that works nicely to detected them efficiently. So, I suggest to use the same techniques for your cases. You could see two of our related manuscripts already published...
It will give you the results of unspecific gene out of your target. So you should not consider that results for interpreting your gene. It could also give double peaks in the amplification curves of qPCR or very long tail of the amplified curve.
Thank you Ujjal Kumar Nath
You mean that the qPCR assay will work when even several nucleotides of the probe bind the untargeted sequences (for example, only 5 first nucleotides of the probe that contain reporter ) and a part of the forward primers bind the intargeted sequences. Is that correct?
In my opinion, qPCR assay works once totally probe (from reporter to quenche) bind the sequnece at the position that is between forward and reverse primer.
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