《分子生物学分子生物学 (31).pdf》由会员分享，可在线阅读，更多相关《分子生物学分子生物学 (31).pdf（18页珍藏版）》请在taowenge.com淘文阁网|工程机械CAD图纸|机械工程制图|CAD装配图下载|SolidWorks_CaTia_CAD_UG_PROE_设计图分享下载上搜索。
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
6、rs of primers that can simultaneously amplifymultiple regions within the same gene or acrossthe entire genome.Singleplex PCR vs Multiplex PCRMultiplex PCR is widely used ingeneticdiseases diagnose and forensic identification.12.3.4 Multiplex PCRMain Steps of Sorensic Identification Using Multiplex P
7、CR 12.3.5 PCR-ASOPCR-ASO technologyuses PCR to amplify thetarget DNA first,and then uses ASO probe to performdot blot hybridization.Compared with ordinary dot blothybridization,it has higher specificity and sensitivity,and is widely used in gene mutations and pathogenicmicroorganism detection.PCRHyb
8、ridization with ASO probesAccording to whether the 3 end of theprimer is complementaryto the template,two allele specific PCR primers are designed.The mutant or normal allele will be recognizedby the allele specific primers.12.3.6 AS-PCR（allele specific PCR）12.3.7 PCR-RFLPIt uses PCR to specifically
9、 amplify a genefragment,which contains at list one cleavagesite of a restriction endonuclease.PCR productsare then subject to endonuclease digestion.Though analyzing changes in length andnumber of the digested DNA fragments byelectrophoresis,gene mutaions are finallyidentified.5-GAATTC-33-CTTAAG-5Se
10、quence Change of the EcoR Recognition SiteABPCR productsC5-GGATTC-33-CCTAAG-5N:Normal homozygote M:mutant homozygote H:heterozygoteNHMEcoRdigestionElectrophoresisseparation12.3.7 PCR-RFLP12.3.8 DNA SequencingRepresentative results of Sanger Automated SequencingNon-invasive prenatal diagnosis Using Next Generation Sequencing12.3.8 DNA SequencingThank you!