properties-of-DNA(07-2)改.pptVIP

* * * * * * * * * * In a real time qPCR assay, the probe hybridizes to a target sequence during the annealing step of PCR and it will remain annealed during the extension. The probe is partially displaced by the polymerase during extension. Next, the probe is cleaved by 5’- 3’ nuclease activity of the polymerase, which allows the now free reporter molecule to absorb light and emit fluorescence at its characteristic wavelength. PCR Targets The number of bases in the targets can vary. TTAAGGCTCGA . . . . AATTGGTTAA The . . . . Represents the middle DNA sequence, and does not have to be known to replicate it. PCR Denaturing Denaturing is the first step in PCR, in which the DNA strands are separated by heating to 95°C. PCR Primers Primers range from 15 to 30 nucleotides, are single-stranded, and are used for the complementary building blocks of the target sequence. PCR Primers A primer for each target sequence on the end of your DNA is needed. This allows both strands to be copied simultaneously in both directions. PCR Primers The primers are added in excess so they will bind to the target DNA instead of the two strands binding back to each other. PCR Annealing Annealing is the process of allowing two sequences of DNA to form hydrogen bonds.The annealing of the target sequences and primers is done by cooling the DNA to 55°C. PCR Taq DNA Polymerase Taq stands for Thermus aquaticus, which is a microbe found in 70～75℃ hot Springs in Yellow Stone National Forest. PCR Taq DNA Polymerase Taq produces an enzyme called DNA polymerase, that amplifies the DNA from the primers by the polymerase chain reaction, in the presence of Mg. PCR Cycles PCR Cycles PCR Cycles PCR Cycles PCR Cycles PCR Cycles Review Denaturalization: 94°- 95°C Primer Annealing: 55°- 65°C Extension of DNA: 72° Number of Cycles: 25-40 PCR Formula 循环数 拷贝数 目标条数 非目标条数 1 2 0 4 2 4 2 6 3 8 8 8 4 16 22 10 5 32 52 12 6 64 114 14 n(n=1,2,3.) 2^n 2*2^n-2n-2 2n+2 PCR Requirements Pro