for investigate the mutation in Helicobacter pylori 23srRNA gene, the DNA samples of the bacterium were analyzed by real time pcr taqman probe.The reaction was carried out in two steep conditions under the following conditions: the reaction was performed in volume 12 with the following details:( Master MIX: 6 λ/Primer F: 0.4 λ/Primer R: 0.4 λ/Prob: 0.2 λ/H2O: 3 λ/DMSO:1 λ/DNA:1 λ)
(Hold:95c 10 min/Cycle 1: 95c 15 s/Cycle 2: 58c20 s/Cycle3: 72c 20s / Number of cycles:40) on Rotor-Gen3000 device). Annealing temperature was setted by PCR (.two double-labeled probes used are(1-FAM-TAMRA and 2-HEX-BHQ1)(Taq copenhang –denmark)( Tm company suggested are 37c and 44 c)
Results read on device that are unacceptable and illogical signal and no fluorescent light in the fam and hex channels as shown in the attached pictures(1)
The Real time pcr product was runed on gel 8% polyacrylamide and the 87bp target gene fragment was observed in all samples.these results indicate that the reaction occurs and the product is produced but the probe is not connected to target position, because the fluorescent light and the signal are not logical due to one of the following conditions1 - either the probe is connected but the reporter or quencher has a problem or 2 - the probe is flawless but unable to connect to the target site
To determine whether the probes have fluorescents was used from each of the probes 1 λ, from the main stock of probes with a concentration of 100 μmol with 1 λ (DNase I ) in the negative control samples.(The aim was to artificially break the probe by the DNase I and remove the reporter from the quencher to emit light.)
This time Fluorescent light was observed in negative control samples containing dnase, which indicates the presence of fluorescent probe and now likely the cause of the problem is that the probe not to bind to the region.
Why don't probes attach to the sequence? What solution do you suggest?
- You have trouble shooted well
- I can only assume that your probe does not bind to your target region because their is a sequence difference in your sample targets that is stopping it binding ?
- Are you certain of the target region sequence ?
- If not clone and sequence to be sure. That would be my advice
- Your own investigations have ruled out the usual technical artifacts pointing to something more fundamental like a target mutation/polymorphism
Hello Razieh
You did well troubleshooting! First of all you said a 2-step-reaction but your program was 3-step. Maybe with 2-step would help. The only possibility came to my mind is the sequence of your probe or any mutations in 23srRNA fragments.
hi.thanks for answer. two-step method was also used the results were similar than. at first two-steep selected on the device were then manually added to a step ( to separate the annealing cycle from extension cycle ) because It was better band on gel. 2- I have separate probe for no-mutation mode that if it has no mutation sequence it must be attached but it doesn't connect .
Use gradient with real time PCR machine.
If you will not get answer, clearly your prob synthesis had problem.
Regards.
- You have trouble shooted well
- I can only assume that your probe does not bind to your target region because their is a sequence difference in your sample targets that is stopping it binding ?
- Are you certain of the target region sequence ?
- If not clone and sequence to be sure. That would be my advice
- Your own investigations have ruled out the usual technical artifacts pointing to something more fundamental like a target mutation/polymorphism
Alireza Mordadi Thank you . I used gradient for the target gene with PCR and selected 58 c.
Laurence Stuart Dawkins-Hall Thank you very much .
I checked the forward and Revers primer sequence and probe position with Gene Runner software. )the photo is attached(,forward marked with blue , reverse with Brown and Probe with Pink (for No Mutation Mode)
and another probe that has not been identified is a different base with this sequence, so I'm sure of the target gene sequence and the ordered sequence for my probes.now what is the cause? does the probe sequence have a problem? since the probe binding temperature is different from the primer binding temperature is the reason for the lack of connection due to the annealing temperature? we set the temperature for the binding of the primers (58c) and manufacturer's suggested temperature for probes are 37c and 44 c!!??
Ali Javadmanesh hi.thanks for answer. two-step method was also used the results were similar than. at first two-steep selected on the device were then manually added to a step ( to separate the annealing cycle from extension cycle ) because It was better band on gel. 2- I have separate probe for no-mutation mode that if it has no mutation sequence it must be attached but it doesn't connect .
Are primers and probes specifically designed for Helicobacter pylori 23srRNA gene? Try to add DNA 2uL, water to lead volume 18uL. Try with dilution series to test 10^6 gene copies.
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