分子生物学分子生物学 (17).pdf

Chapter 7Basic Methods of Gene Structure and Expression Analysis7.3 Real7.3 Real-time time quantitative PCRquantitative PCR（RealReal-time PCRtime PCR）RealReal-time quantitative PCRtime quantitative PCR（RealReal-time time PCRPCR）RealReal-timetimePCRPCRapplicationapplication:ForForquantitativequantitative analysisanalysis ofof genegene DNADNA copycopynumbernumber andand mRNAmRNA expressionexpression.PCR technology for realPCR technology for real-time time quantification of products through realquantification of products through real-time time quantitative monitoring and fluorescence quantitative monitoring and fluorescence detection system to monitor accumulated detection system to monitor accumulated fluorescence intensity.fluorescence intensity.Fluorescent dyes1.1.Fluorescent labeling methodFluorescent labeling method Hydrolysis probeHydrolysis probeThe 5 end of the oligonucleotide probe is The 5 end of the oligonucleotide probe is labeled with a fluorescent reporter labeled with a fluorescent reporter groupgroup(reporter)(reporter),and the 3 end is labeled with,and the 3 end is labeled with a fluorescence quenching groupa fluorescence quenching group(quencher)(quencher).TaqManTaqMan Probe Probe The TaqMan hydrolysis probe has about 20 to 30 nucleotides and is completely complementary to the target gene.A fluorescent reporter group is labeled at the 5 end and a fluorescent quencher group is labeled at the 3 end.TaqManTaqMan Probe Probe Molecular Beacon ProbeMolecular Beacon ProbeTheThe probeprobe hashas a a hairpinhairpin structure,structure,thethe 5 5 endendisis labeledlabeled withwith a a fluorescentfluorescent reporterreporter group,group,andandthethe 3 3 endend isis labeledlabeled withwith a a quencherquencher groupgroup.When the probe hybridizes with the target sequence,the reporter fluorescent dye is separated from the quenching dye and emits fluorescence.CycleThe amount of end point DNA is the amount amplified by PCR.There is some distortion.The quantitative reproducibility of the starting point is good.End point quantitative error is large.2.Principle of calculationCT value:the number of cycles corresponding to the inflection point from the baseline to the exponential growth.Standard curve:CTvalue plotted against DNA 013Fluorescence quantitative PCR instrumentThank You!