分子生物学分子生物学 (18).pdf

Chapter 7Basic Methods of Gene Structure and Expression Analysis7.4 Nucleic Acid Hybridization Nucleic acid molecule hybridization refers Nucleic acid molecule hybridization refers to the process in which two complementary to the process in which two complementary singlesingle-stranded nucleic acids form double stranded nucleic acids form double strands under certain conditions according to strands under certain conditions according to the principle of base pairing.the principle of base pairing.7.4.7.4.1 Principle of nucleic acid 1 Principle of nucleic acid molecular hybridization molecular hybridization Nucleic acid molecule hybridization is essentially the renaturation process of two single-stranded strands with complementary sequences after denaturation of double-stranded DNA or RNA with secondary structure.1denaturation2renaturation3hybridizationVarious physical methods are used Various physical methods are used to transfer the biomacromolecules in to transfer the biomacromolecules in electrophoresis to various membranes electrophoresis to various membranes such as nylon membrane and NC membrane such as nylon membrane and NC membrane to make them into solid phase to make them into solid phase molecules.molecules.(1)Imprinting technology(1)Imprinting technologyA polynucleotide chain of a known sequence labeled A polynucleotide chain of a known sequence labeled with a radionuclide,biotin,or fluorescein dye at its with a radionuclide,biotin,or fluorescein dye at its end or full chain is called a probe.end or full chain is called a probe.(2)Probe labeling technology(2)Probe labeling technologyRenaturationRNADNAThe labeled singleThe labeled single-stranded stranded nucleic acid probe partially nucleic acid probe partially complements the target singlecomplements the target single-stranded stranded DNA or RNA to be detected to form a DNA or RNA to be detected to form a hybrid doublehybrid double-strand.strand.2.Southern blotting2.Southern blottingThe process of binding the DNA fragments The process of binding the DNA fragments separated by electrophoresis to a certain solid separated by electrophoresis to a certain solid support,and then performing hybridization with support,and then performing hybridization with the nucleic acid probe labeled in the liquid the nucleic acid probe labeled in the liquid phase.phase.Used for DNA qualitative and Used for DNA qualitative and quantitative analysis.quantitative analysis.2020/5/3091.Preparation of genomic DNA1.Preparation of genomic DNA2.Digest genomic DNA with restriction endonucleases2.Digest genomic DNA with restriction endonucleases3.Separation of DNA digested fragments by agarose 3.Separation of DNA digested fragments by agarose gel electrophoresisgel electrophoresis4.Gel pretreatment4.Gel pretreatment5.Southern blot transfer5.Southern blot transfer6.Hybridization of labeled nucleic acid probes and 6.Hybridization of labeled nucleic acid probes and probes with DNA fragmentsprobes with DNA fragments7.Hybridization result display7.Hybridization result displayBasic process of Southern blot hybridizationBasic process of Southern blot hybridizationSeparation of DNA digested fragments by agarose gel electrophoresisSeparation of DNA digested fragments by agarose gel electrophoresisSouthern blot transferSouthern blot transferHybridization results show Hybridization results show Digestion analysis of cloned Digestion analysis of cloned genesgenesQualitative and quantitative Qualitative and quantitative analysis of genomic genesanalysis of genomic genesGene mutation analysisGene mutation analysisRestriction fragment length Restriction fragment length polymorphism analysis polymorphism analysis DNA can be analyzed.DNA can be analyzed.Southern blot hybridization Southern blot hybridization applicationsapplications3.3.Northern blottingNorthern blottingAfter denaturing and electrophoretic After denaturing and electrophoretic separation of RNA,and transferred to a separation of RNA,and transferred to a solid support,the hybridization solid support,the hybridization reaction was used to identify the reaction was used to identify the content and size of specific mRNA content and size of specific mRNA molecules.molecules.For qualitative and quantitative For qualitative and quantitative analysis of mRNA.analysis of mRNA.HousekeepingHousekeeping genegenetarget genetarget gene0d 2d 4d 6d 8dNorthern blotting to detect gene expressionNorthern blotting to detect gene expression4.Dot and slot blot hybridization4.Dot and slot blot hybridizationRNA or DNA is denatured and RNA or DNA is denatured and directly spotted on nitrocellulose directly spotted on nitrocellulose membrane or nylon membrane for membrane or nylon membrane for qualitative and quantitative study qualitative and quantitative study of specific genes and their of specific genes and their expression in the genome.expression in the genome.5.5.In situIn situhybridizationhybridizationThe labeled nucleic acid probe is used to The labeled nucleic acid probe is used to hybridize with the complementary sequence of the hybridize with the complementary sequence of the nucleic acid to be tested in the cell,and the nucleic acid to be tested in the cell,and the nucleic acid can be characterized,positioned and nucleic acid can be characterized,positioned and relatively quantitatively analyzed.relatively quantitatively analyzed.DNA DNA Chip and MicroarrayChip and MicroarrayThe array on the gene chip is composed The array on the gene chip is composed of regularly arranged samples such as of regularly arranged samples such as cDNA or oligonucleotides.cDNA or oligonucleotides.cDNA microarraycDNA microarray Oligonucleotide microarrayOligonucleotide microarrayC CB BA ACy5Cy5-labeled cDNAlabeled cDNACy3Cy3-labeled cDNAlabeled cDNAC CB BA AC CB BA AC CB BA AC CB BA AC CB BA AC CB BA Asample 1sample 1sample 2sample 2Cy5/Cy3 Cy5/Cy3 fluorescent labelfluorescent labelRNA extractionRNA extractionCompetitive Competitive hybridization shows hybridization shows differences in gene differences in gene expressionexpressionSchematic diagram of competitive Schematic diagram of competitive hybridization of two sampleshybridization of two samplesPrinciple of Gene Principle of Gene Expression Profile ChipExpression Profile ChipThe main technical process of gene The main technical process of gene expression profile chipexpression profile chip1.Sample fluorescent 1.Sample fluorescent labellabel2.Chip preparation2.Chip preparation3.Chip hybridization3.Chip hybridization4.Chip washing and 4.Chip washing and scanningscanning5.Scanned image 5.Scanned image analysisanalysisexperimental designexperimental designGene chip experimentGene chip experimentImage analysisImage analysisGene expression profile chip Gene expression profile chip research application ideasresearch application ideasBiological issues are raisedBiological issues are raised.Gene expression analysisGene expression analysis.Sample classification and Sample classification and experimental predictionexperimental predictionSome genes are Some genes are abnormal or differentabnormal or differentBiological confirmation and Biological confirmation and interpretation analysisinterpretation analysisNext experimental designNext experimental designStandardization of Standardization of resultsresultsTheThe cDNAcDNA chipchip cancan quantitativelyquantitativelymonitormonitor thethe expressionexpression levellevel ofof a a largelargenumbernumber ofof genes,genes,explainexplain thethe functionfunction ofofgenes,genes,exploreexplore thethe causescauses andand mechanismsmechanismsofof diseases,diseases,andand discoverdiscover targettarget genesgenesforfor diagnosisdiagnosis andand treatmenttreatment.Real time RTReal time RT-PCR verification.PCR verification.Bioinformatics analysis of gene Bioinformatics analysis of gene expression data.expression data.Application of cDNA chip in medicineApplication of cDNA chip in medicine2020/5/30Section 5 Western BlotSection 5 Western BlotWesternWestern blotblot principleprinciple isis similarsimilar totonucleicnucleic acidacid moleculemolecule hybridization,hybridization,exceptexcept thatthat thethe antibodyantibody moleculemoleculeconjugatedconjugated withwith thethe labellabel isis usedused asas a aprobeprobe toto detectdetect thethe proteinprotein moleculemoleculetransferredtransferred toto thethe solidsolid supportsupport.AntibodyAntibodyEnzymeEnzymeSubstrateSubstratePVDF membraneProteinProteinDetectable productsDetectable productsProtein sample preparationProtein sample preparationSDSSDS-PAGE separationPAGE separationProtein transmembraneProtein transmembraneLabeled antibody hybridizes Labeled antibody hybridizes to protein antigens on the to protein antigens on the membranemembraneDetect and analyze marker Detect and analyze marker signalssignalsWestern blot program:Western blot program:Thank You!