荧光定量原理与应用第三版.ppt

* 10^6 copies * 10^4 copies * And finally 10^2 copies If we look at these profiles they seem to have the same curvature at least during the early amplification phases. We can also notice that a regular dilution series appear at a regular interval. * To these profiles, We set a benchmark, the Threshold line (T). This is an arbitrary line that can vary quite a bit. It does have one restriction, it must be set during the exponentially growth phase of the amplifications. From this threshold, we collect CT values. These values simply represent the number of cycles the sample required to reach the amount of product (fluorescence) marked by the Threshold line. * If you know the amount of target DNA present in the samples at the start… These CT’s can be plotted as a function of starting quantity and generate a standard curve. * Any unknowns that want to be quantified are run concurrently with our standards. The CT of the unknown is collected……. * …. And plotted against the standard curve. This gives us an accurate determination of the number of copies of our target DNA in our unknowns. A simple analogy: QPCR is similar to running a protein assay such as a Bradford or a Lowry. The difference is that instead of using a genetic standard such as BSA, QPCR requires the gene itself to be used as a standard. What we are actually doing is determining the efficiency of our assay in order to determine relative to a standard, what is I the unknowns. * End point analysis does not accurately represent real initial values. At 40 cycles, samples at 10^8, 10^6 and 10^4 copies have about the same amount while 10^2 copies has about 30% less. Even if we stop the analysis at 20 cycles, sample at 10^6 copies seems to be 50% of 10^8 copies. The 10^4 and 10^2 copies seen to have nothing in them. Again qualitative yes, Quantitative no. * Essentially, if we wait until the end of the reaction, uniformity and reproducibility can vary enormously. On the other hand, if we look at our assay