BioRad定量PCR说明书课件.ppt

* If you start with x copies of template, TaqMan liberates x reporters in the first cycle repeat. In the second cycle repeat TaqMan results in a total of 3x reporters in solution In the third cycle repeat, TaqMan results in a total of 7x reporters in solution In the 25th cycle repeat, TaqMan results in a total of 33,554,431x reporters in solution * Describe the beacon structure here * * * * Tthere are a few more constraints on probe design vs. primer design: Higher annealing temperature than potential primers. This ensures that the probes anneal to the target sequence BEFORE the primers anneal. Think of the reaction dynamics as the PCR sample goes from the denaturation step at 95 degrees down to the annealing temperature of 55 degrees or so… The restriction on guanidine residues at the 5’ end of the probe prevents undesired signal quenching, particularly for fluorescein labeled probes. * SYBR is cheap and effective but one cannot multiplex with it. If one has no reason to multiplex then there is often no reason to use one of the more expensive chemistries. Cleavage probes give you the ability to multiplex but do not have the high degree of specificity of molecular beacons. The additional specificity of the probe based chemistries is not as much of an advantage as one might think. Primers dictate the kinetics of the reaction. Adding an internal oligo like a cleavage probe to an assay that is not specific has limited benefits. * Practice with the animation so you understand the timing of this slide (if you haven’t used it before) - ask the audience to participate in this, it is a good way for you to determine the different comfort levels your audience has with the Ct concept. See next slide for relationship between dilution series and threshold cycle values. * Further elaboration on the exponential nature of PCR. The mathematic expression that describes the process is shown. Some basic implications are listed. * Plot of Ct versus copy number.