Hi All,
I am trying to perform melt curve analysis for a single gene using Taqman probe. I performed asymmetric QPCR on StepOne Plus. Even though I got the melt curve peaks, they are not as distinct from negative control (Tm 79.6 for NC and 82.4 for Test). In SYBR however, I usually get a huge Tm difference between NC and test.
It was a test experiment and I plan to use molecular Beacons. I was was wondering if the less Tm difference is due to TaqMan probe which is a hydrolysis probe?
Does melt curve analysis work well on TaqMan?
I appreciate your help!
You need to use an intercalating dyes or FRET probes...hydrolysis probes cannot be used for meelting analysis. Probably you also need to review some theory about qPCR and dyes. I recommend you https://www.gene-quantification.de/
best regards
Dear Renu, you can use SYBRGreen or similar dyes for gene expression analysis and then you can perform melting curve analysis.
You can use TaqMan probes for melting. I'am using such probes for SNP detection. What is your negative control?
Dear Konstantin,
Thank you. I am using sloppy molecular Beacons for SNP detection. I could optimize my multiplex experiments.
Thank you all for your inputs.
Renu I think technologies are mixed up here.
Sybr green or similar systems are good for melt curve analysis. The idea is that double stranded DNS sequesters a dye that could either be quenched or hyperfluorescent, with the opposite taking place upon melting.
TaqMan is not based on the principle of a Dye being sequestered by double stranded DNA. They have an internal probe that gets unquenched when polymerase runs down the template strand from the primer.
Get someone on the phone at TaqMan. They help. In theory, I do not think theer is a brick wall between using internal probe and also having a useful intercalating dye.
It's just that all of that would have to be in the reaction.
I would like to have melt curve analysis following qPCR with TaqMan, but I do not think there current systems do this.
Agree with Neal. Taqman probe can't be used for melting curve analysis. Only when using Sybr-Green or similar intercalating dyes need to do melting curve analysis as Sybr-green is non-specific bind to double stranded DNA. Taqman is specific bind to template sequence. Taqman’s signal is only collected during real-time PCR amplification with polymerase , not after.
Honghua Hu just look here https://www.mrcholland.com/technology/melt-assays if you have doubts how taqman probes work for melting.
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