生理所黄阿敏精.ppt

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1、生理所黄阿敏第1页，本讲稿共40页Comparison of Southern,Northern,and Western analyses of Gene X第2页，本讲稿共40页Southern hybridizationnFirst described by E.M.Southern in 1975.nApplications of Southern hybridizationnRFLPs,VNTRs and DNA fingerprintingnChecking of the gene knockout micenThe flow chart of Southern hybridizat

2、ion第3页，本讲稿共40页Southern hybridizationTransfer buffer第4页，本讲稿共40页Detection of an RFLP by Southern blotting第5页，本讲稿共40页Detection of the sickle-cell globin gene by Southern blotting第6页，本讲稿共40页Checking of the gene knockout mice第7页，本讲稿共40页Flow chart of Southern hybridizationPreparing the samples and running

3、 the gelSouthern transferProbe preparationPrehybridizationHybridizationPost-hybridization washingSignal detectionIsotopeNon-isotope第8页，本讲稿共40页Preparing the samples and running the gelnDigest 10 pg to 10 g of desired DNA samples to completion.nPrepare an agarose gel,load samples(remember marker),and

4、electrophorese.nStain gel ethidium bromide solution(0.5 g/ml).nPhotograph gel(with ruler).第9页，本讲稿共40页Critical parameters(I)nNote the complexity of DNAnGenomic DNAnA single-copy of mammalian gene,3 Kb average in length 10 g x 3 Kb/3 x 106 Kb=10 g x 1/106=10 pgnPlasmid DNA or PCR products 0.1 g of a 3

5、 Kb plasmid DNA 100 ng第10页，本讲稿共40页 Gel treatmentnAcid treatment n0.2 N HCl solution nDenaturationnNaOH solutionnNeutralizationnTris-Cl buffer(pH8.0)第11页，本讲稿共40页Southern transfernMeasure gel and set up transfer assembly:nWick in tray with 20 x SSCnGelnNitrocellulose or Nylon filters(soaked in H2O and

6、 20 x SSC)n3MM Whatman filter papernPaper towelsnWeight第12页，本讲稿共40页After Southern transfernDissemble transfer pyramid and rinse nitrocellulose in 2x SSCnBake nitrocellulose at 80C for 2 hr or UV-crosslink Nylon membrane for seconds 第13页，本讲稿共40页Preparation of probesnSynthesis of uniformly labeled dou

7、ble-stranded DNA probesnPreparation of single-stranded probesnLabeling the 5 and 3 termini of DNA第14页，本讲稿共40页Synthesis of double-stranded DNA probes-Nick translation of DNA-Labeled DNA probes using random oligonucleotide primers第15页，本讲稿共40页Nick translation第16页，本讲稿共40页Preparation of single-stranded p

8、robesnSynthesis of single-stranded DNA probes using bacteriophage M13 vectors.nSynthesis of RNA probes by in vitro transcription by bacteriophage DNA-dependent RNA polymerase.第17页，本讲稿共40页In vitro transcription第18页，本讲稿共40页nLabeling the 3 termini of double-stranded DNA using the Klenow fragment of E.c

9、oli DNA polymerase I.(lack of 5 3 exonuclease activity)nLabeling the 3 termini of double-stranded DNA using bacteriophage T4 DNA polymerase.nLabeling the 5 termini of DNA with bacteriophage T4 polynucleotide kinase.Labeling the 5 and 3 termini of DNA第19页，本讲稿共40页T4 polynucleotide kinase activity第20页，

10、本讲稿共40页Non-isotope labelingnDigoxigenin-11-dUTP(DIG-dUTP)labeling nDNA labelingnOligonucleotide labelingnRNA labeling第21页，本讲稿共40页PCR Labeling,Random Primed Labeling,and RNA Labeling第22页，本讲稿共40页Prehybridization nAdd prehybridization solution and prehybridize at hybridization temperature for 2-4 hr 第2

11、3页，本讲稿共40页Hybridization nRemove prehybridization solution and add hybridization solutionnAdd 500,000 cpm of the probe/ml hybridization solution.nHybridize overnight at appropriate temperature.第24页，本讲稿共40页Post-hybridization washing n Wash twice,15 min each,in 1x SSC,0.1%SDS at room temperature.nWash

12、twice,15 min each,in 0.25x SSC,0.1%SDS at hybridization temp第25页，本讲稿共40页Critical parameters(II)nHomology between the probe and the sequences being detectednTm=81+16.6(log Ci)+0.4%(G+C)-0.6(%formamide)-600/n-1.5(%mismatch)nFactors can be changed:nHybridization temp.nWashing temp.nSalt concentration d

13、uring washingHigh temp.,low salt:high stringencyLow temp.,high salt:low stringencynIf 50%formamide is usedn42 oC for 95 100%homologyn37 oC for 90 95%homologyn32 oC for 85 90%homology第26页，本讲稿共40页Comparison of nitrocellulose and nylon membranesNC Nylon Hydrophobic binding Covalent binding Fragile Dura

14、ble Probe length 200 300 bp 0.05%of the message),total cellular RNA can be used.If the mRNA species of interest is relatively rare,however,it is advisable to use poly(A)+RNA.nIncubate 15 min at 55C第33页，本讲稿共40页Running the RNA gelnAdd 10 l formaldehyde loading buffer to each sample and load gel.Run ge

15、l at 100 to 120 V for 3hr.nRemove gel from the running tank and rinse several times in water.Place gel in 10 x SSC for 45 min.nDo not need post-transferring gel treatment第34页，本讲稿共40页An example of Northern blottingNorthern blotRNA gel 28 S 18 S第35页，本讲稿共40页Western blotting,or immunoblottingTechnique f

16、or detecting specific proteins separated by electrophoresis by use of labeled antibodies.第36页，本讲稿共40页Flow chart of Western blottingElectrophoresing the protein sampleAssembling the Western blot sandwichTransferring proteins from gel to nitrocellulose paperStaining of transferred proteinsBlocking non

17、specific antibody sites on the nitrocellulose paperProbing electroblotted proteins with primary antibodyWashing away nonspecifically bound primary antibodyDetecting bound antibody by horseradish peroxidase-anti-Ig conjugate and formation of a diaminobenzidine(DAB)precipitatePhotographing the immunob

18、lot第37页，本讲稿共40页SDS polyacrylamide-gel electrophoresis(SDS-PAGE)第38页，本讲稿共40页Analysis of protein samples by SDS polyacrylamide-gel electrophoresis and Western blottingProtein bands detected by specific antibodySDS-PAGEWestern blot第39页，本讲稿共40页Comparison of Southern,Northern,and Western blotting techniques第40页，本讲稿共40页