分子生物学分子生物学 (31).pdf
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1、Chapter 12 Gene Diagnosis(PCR and DNA Sequencing)12.3 Molecular Biology Techniques Commonly Used in Gene Diagnosis12.3.1 Common PCR Derived Techniques in gene diagnosisSpecific Amplification of target DNA by PCRRT-PCRReal-Time Fluorescence Quantitative PCRMultiplex PCRAS-PCRPCR-ASO PCR-RFLP12.3.1 Co
2、mmon PCR Derived Techniques in gene diagnosis12.3.2 RT-PCR(Reverse Transcription-PCR)RT-PCR,reverse transcription PCR,is a fastand sensitive method for RNA detection.DuringRT-PCR,RNA is reversely transcripted to cDNAfirst,and then cDNA is used as a template for PCRreaction.PCRRT-PCRTarget geneRefere
3、nce geneRT-PCR can be used to detect the presenceof target RNAs in the sample,or quantifyRNAs relative to the reference gene.Representative Results of RT-PCR12.3.2 RT-PCR(Reverse Transcription-PCR)12.3.3 Real-time fluorescent quantitative PCRReal-time fluorescent quantitative PCR(qPCR),or real-time
4、PCR,is a fast and quantitative methodfor target nucleic acids.During Real-time fluorescentquantitative,fluorescent dyes or probes are added tothe PCR system to detect PCR amplification in realtime.Ct0 10 20 30 400 10 20 30 401 2 3 4 5 6 7 81 2 3 4 5 6 7 8Log(N0)Amplification Curve and Standard Curve
5、 of qPCR12.3.3 Real-time fluorescent quantitative PCRReal-time fluorescence quantitative PCRtechnologyhas been widely used in clinicalpathogenicmicroorganismdetectionandgenemutation diagnosis.12.3.3 Real-time fluorescent quantitative PCR12.3.4 Multiplex PCRA multiplex PCR system contains multiplepai
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