"Normal qPCR" is a curious term - as that must be defined further, but, for these purposes, I'll assume you meant SYBR Green-based (or EVA-Green-based) qPCR as being "normal qPCR," since hydrolysis probe-based qPCR is indeed a different beast.
A general synopsis involving a hydrolysis probe (of which TaqMan is a variety) and two accompanying primers (FWD and REV) might be described as follows:
Generally one wishes to design the probe to have a Tm of ~70C while the two primers that usually accompany it are designed to have Tms of ~60C. This is so that the probe can hybridize to its target sequence first (it anneals to its target sequence before the primers do). The PCR cannot start when the probe is in place since it is blocked on the 3' end. The 5'-end of the probe has a fluor (e.g., 6-FAM, TET, JOE, HEX, etc., while the 3'-end has a quencher of some kind e.g., TAMRA, BHQ, MGB-NFQ etc. which also prevents the probe from initiating the reaction from the probe's 3' end). If the primers laid down first here, the reaction would be initiated but no reporting would occur - so, that is why it is important that the probe's Tm is 5 to 10 degrees higher than the primer Tms.
Once the cycling temperature reaches ~60C (50% of the probe is already in place at ~70C; even more probe is annealed at 60C), 50% of the primers are annealed and the PCR is initiated. The primer that lands on the same strand the probe has already landed on, allows Taq (or another similarly effective DNA-dependent DNA polymerase) to start extending from the 3' end of that primer toward the probe's 5' end.
Next, the unique 5'-exonuclease activity of the 'Taq' enzyme, degrades the probe (first clipping about 3 nucleotides into the 5'-end of the probe) at the same time it extends the amplicon in 5' to 3' direction from the 3'-end of the associated primer. Degradation of the probe liberates the 5'-fluor end from the probe and releases it from the proximal quenching effect of the 3'-quenching molecule, allowing the fluor to report/fluoresce more loudly so that the reaction can be detected by the machine in real time. If the probe is not degraded, no signal will occur.
This is a very basic description, but it may hold some helpful details (such as remembering that the particular Taq or other similar enzyme used in a 'hydrolysis probe 'assay, is also understood to harbor this unique 5'-exonuclease activity; without which, the probe would not be degraded, and the reaction would not report a measureable signal)...
Hi!
TaqMan is named after the famous old game PacMan. I used several times for SNP genotyping, and it consists of 2 probes especially design to anneal during RealTime-PCR with either one of your SNP's alleles or the other. Thus, the 2 probes must be equal in their sequence (in order to be stable enough) except for the nucleotide which will anneal with your specific SNP - so each probe will anneal specifically with one allele or the other. Moreover, each one of these probes include a specific flourophore (a molecule which will display a certain fluorescence) in one end, and a quencher on the other - which will by now inhibit that fluorescence. Then, for every run in your PCR, one of your probes - or both, in case your sample is heterozygous for that SNP- will anneal with your sample, and when the Taq Polymerase comes around to replicate that region (just like the big yellow round face in PacMan) the probe will be torn apart, thus losing the quencher effect on the fluorophore, and displaying flourescence. Such fluorescence will be read by your machine, and give a diagnostic based on wich Fluorophore (VIC or FAM) is assigned to each allele.
Hope this helps!
He's a little video in addition to Guillermo's explanation.
https://www.youtube.com/watch?v=DNsRiFT3jBs
"Normal qPCR" is a curious term - as that must be defined further, but, for these purposes, I'll assume you meant SYBR Green-based (or EVA-Green-based) qPCR as being "normal qPCR," since hydrolysis probe-based qPCR is indeed a different beast.
A general synopsis involving a hydrolysis probe (of which TaqMan is a variety) and two accompanying primers (FWD and REV) might be described as follows:
Generally one wishes to design the probe to have a Tm of ~70C while the two primers that usually accompany it are designed to have Tms of ~60C. This is so that the probe can hybridize to its target sequence first (it anneals to its target sequence before the primers do). The PCR cannot start when the probe is in place since it is blocked on the 3' end. The 5'-end of the probe has a fluor (e.g., 6-FAM, TET, JOE, HEX, etc., while the 3'-end has a quencher of some kind e.g., TAMRA, BHQ, MGB-NFQ etc. which also prevents the probe from initiating the reaction from the probe's 3' end). If the primers laid down first here, the reaction would be initiated but no reporting would occur - so, that is why it is important that the probe's Tm is 5 to 10 degrees higher than the primer Tms.
Once the cycling temperature reaches ~60C (50% of the probe is already in place at ~70C; even more probe is annealed at 60C), 50% of the primers are annealed and the PCR is initiated. The primer that lands on the same strand the probe has already landed on, allows Taq (or another similarly effective DNA-dependent DNA polymerase) to start extending from the 3' end of that primer toward the probe's 5' end.
Next, the unique 5'-exonuclease activity of the 'Taq' enzyme, degrades the probe (first clipping about 3 nucleotides into the 5'-end of the probe) at the same time it extends the amplicon in 5' to 3' direction from the 3'-end of the associated primer. Degradation of the probe liberates the 5'-fluor end from the probe and releases it from the proximal quenching effect of the 3'-quenching molecule, allowing the fluor to report/fluoresce more loudly so that the reaction can be detected by the machine in real time. If the probe is not degraded, no signal will occur.
This is a very basic description, but it may hold some helpful details (such as remembering that the particular Taq or other similar enzyme used in a 'hydrolysis probe 'assay, is also understood to harbor this unique 5'-exonuclease activity; without which, the probe would not be degraded, and the reaction would not report a measureable signal)...
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