PCR技术的新进展和应用.ppt

Who will be the next Kary Mullis? Khorana HG. 1971; Journal of Molecular Biology, 56:341: DNA PCR Mullis K. 1983; PCR Telling GC. 2001; Protein-based PCR, Nature Medicine, 2001 Jul;7(7):778-9 Who? Protein PCR 谢谢观看！ 2020 One question is how one uses real-time PCR to quantitate the amounts of DNA or cDNA. The first two calculation methods for real-time PCR results that we are going to focus on are equivalent to the calculations that one usually does when one does a Northern. This slide shows a virtual Northern with two lanes, one with RNA from control cells, the other with RNA from the experimental sample (eg drug treated cells). For the sake of argument, let’s say that there is 10x the amount of signal in the experimental sample compared to the control sample for the target gene. This could mean expression of the gene has increased 10-fold, or it could mean that there is 10x as much RNA in the expt lane. To check for this one usally does a so-called ‘loading control’ in which the blot is probed for expression of a gene which does not change (e.g. actin, GAPDH, cyclophilin, RPLP0 mRNAs; ribosomaL RNA). In this case, let’s say that the loading control shows that there is twice as much RNA in the expt lane. Thus the real change in the target gene is 10/2 =5 fold. We can express this in a more general fashion: ratio target gene (experimental/control) = fold change in target gene (expt/control) fold change in reference gene (expt/control) Here is an example of how one could set up a plate if one were using the standard curve method. By clicking on one of the symbols in the top line and then clicking on one of the wells in the plate diagram, one can define whether samples contain a standard (circles) or an unknown (squares) or negative control samples containing water instead of DNA (-) and whether one has single wells containing the same sample or duplicate or triplicate wells (in which case the duplicate or triplicate well are assigned the same