实时荧光定量PCR技术与应用讲义.ppt

* SYBR Green I dye has an increased fluorescent intensity when bound to double stranded DNA. * When sample is denatured, signal drops. When more double stranded DNA is generated, the signal increases proportionally with the amount of dsDNA present. * * How does melt curve work? – After the main amplification run, usually 40 cycles, We set the instrument to collect periodic fluorescent readings of our samples as we slowly ramp the block from 55 to 95 degrees centigrade. Initially signals seem to drop (slow decay) caused by the change in pH of our buffer. Remember that as the temperature of a buffer changes, so does the pH. If there is double stranded DNA, at some point the strands will melt away. When this occurs, the SYBR signal drops sharply as the dye is no longer intercalated. Different products will generally melt at different temperatures dependant on size, salt concentration and GC-AT ratio. A single drop in signal generally represents a single product. Multiple drops represent multiple products in the same well. * The software plots the first derivative which simplifies the analysis. * Probe is added to the PCR mix. Typical probe and primer concentration is around 200 to 300nM. When the temperature is raised, DNA template will denature. Once at 95 degrees, blocking agent will breakdown activating the polymerase. * Upon cooling, the probe and primers will anneal their complementary sequences. The critical parameter is to make sure that the probes anneals before the primers do. Typically, designing the probe to anneal 7 - 10 degrees higher than the primers will do the trick. The advancing polymerase should encounter the probe and hydrolyze it thus releasing a fluor into the mix. If the primers annealing before the probe does, the polymerase will extend the primer quickly and probably prevent probe from annealing. Sometimes, users will run this type of assay and see no amp signal. They will run the sample on an agarose gel and see a band. They may