We have an issue with the Taqman Probe (FAM-BHQ1) based real time detection of target.
What we have been observing is at higher copy numbers >2Crore we are getting very good amplification plots but at lower copy numbers of plasmid instead of a clear S curver amplification plots we are seeing straight lines as shown in the image black cricled plots.
The distance between my Sense primer to Sense probe is 100 bp, total amplicon size 196 bp. Does designing a probe so far from the sense primer has some role to play in sensitivity of detection and straight line plots
no your amplicon size is not a problem, however try to make serial dilution of you samples and check the results
Check whether the template concentration you are using is too dilute. At very low concentration of template, you are likely to observe what you call straight lines (Ref.: https://www.biotechniques.com/multimedia/archive/00008/98245bm09_8067a.pdf )
Hello Arafat, My concern is not the amplicon size which is 196 bp, whether the probe detection would go down if the sense probe position is 100 bp far from the sense primer.?
Is there any co relation between distance between primer and probe to that of sensitivity of detection.
I did come across answers saying it is good to keep 5 bp distance between primer and probe for instant release of the signal, But logically even if it is placed 100 bp far the probe would bind to target itself and gets release only when polymerase comes to the sight right?
So will long distance between primer and probe has any role to play in sensitivity and signal release capacity is my query.
Hello Waseem , Thanks for the link.
I did perform serial dilutions from 20Lakh copies to 2 copies, yes you are right that I might get a straight line for lower dilutions. But the cT values for these straight lines is coming up very early at 22 itself. Generally for lower dilutions like 20000,2000,200 we get 32,36,38 cT values in SyBr Green based assay for the same primers (with out probe) but using SyBr. in this senario we are getting till 2Lakh good plots and suddenly from 20000 we get this straight lines with very less cT difference.
Do you think the 100 bp distance between primer and probe has some role to play with this?
Hey Goutham,
I've never experienced any of it myself. However, if we go by this paper (https://ac.els-cdn.com/S0167701202001136/1-s2.0-S0167701202001136-main.pdf?_tid=04434697-b420-498b-a70b-d47673b2ec00&acdnat=1522843956_657afb25cb6416ddc07eab8ce51b0f95 ), the distance is going to affect your RQ values.
It states, " In general, as either the amplicon size or the primer – probe distance is increased, the DRQ decreases. The effect of primer – probe distance does appear to be more significant as compared to the amplicon size. At a primer –probe distance of
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