分子生物学教学课件Week2modification

1、DNA and RNA isolation, purification, visualization and quantitationGenomic DNA preparation overview Plasmid DNA preparationDNA purificationPhenol extractionEthanol precipitationRNA work1hWhat do we need DNA for?Detect, enumerate, clone genesDetect, enumerate speciesDetect/sequence specific DNA regio

2、nsCreate new DNA “constructs” (recombinant DNA)What about RNA?Which genes are being transcribed?When/where are genes being transcribed?What is the level of transcription?2hcell growthcell harvest and lysisDNA purificationDNA purification: overviewDNA concentration3hBacterial genomic DNA prep: cell e

3、xtractLysis: Detergents Organic solvent Proteases (lysozyme) Heat“cell extract”4hGenomic DNA prep: removing proteins and RNAAdd the enzyme RNase to degrade RNA in the aqueous layer Need to mix gently! (to avoid shearing breakage of the genomic DNA)chloroform5h2 ways to concentrate the genomic DNA70%

4、 final conc.“spooling”Ethanol precipitation6hGenomic DNA prep in plants - how get rid of carbohydrates?CTAB:Cationic detergent(MC 6.61-6.62) (low ionic conditions)N+CH3Br-CH3CH3C16H337hPlasmids: vehicles of recombinant DNABacterial cellgenomic DNAplasmidsNon-chromosomal DNAReplication: independent o

5、f the chromosomeMany copies per cellEasy to isolateEasy to manipulate8hPlasmid purification: alkaline lysisAlkaline conditions denature DNANeutralize: genomic DNA cant renature (plasmids CAN because they never fully separate)9hDNA purification: silica bindingBinding occurs in presence of high salt c

6、oncentration, and is disrupted by elution with water 10hDNA purification: phenol/chloroform extraction1:1 phenol : chloroformor25:24:1 phenol : chloroform : isoamyl alcoholPhenol: denatures proteins, precipitates form at interface between aqueous and organic layerChloroform: increases density of org

7、anic layerIsoamyl alcohol: prevents foaming 11hAqueous volume (at least 200 microliters)Add 2 volumes of phenol:chloroform, mix wellSpin in centrifuge, move aqueous phase to a new tubeRepeat steps 2 and 3 until there is no precipitate at phase interface(extract aqueous layer with 2 volumes of chloro

8、form)Phenol extraction12hEthanol depletes the hydration shell surrounding DNAAllowing cations to interact with the DNA phosphatesReducing repulsive forces between DNA strandsCausing aggregation and precipitation of DNAAqueous volume (example: 200 microliters)- add 22 microliters sodium acetate 3M pH

9、 5.2- add 1 microliter of glycogen (gives a visible pellet)- add 2 volumes (446 microliters) 100% ethanol- mix well, centrifuge at high speed, decant liquid- wash pellet (70% ethanol), dry pellet, dissolve in appropriate volume (then determine DNA concentration)Ethanol precipitation (DNA concentrati

10、on)13hcell growthcell harvest and lysisDNA purificationDNA purification: overviewDNA concentration14hIsolation of RNA - Course reading 11DNA - mRNA - proteinLots of information in mRNA:When is gene expressed?What is timing of gene expression?What is the level of gene expression?(but what does an mRN

11、A measurement really say about expression of the protein?)15hRNA in a typical eukaryotic cell:10-5 micrograms RNA80-85% is ribosomal RNA15-20% is small RNA (tRNA, small nuclear RNAs)About 1-5% is mRNA- variable in size- but usually containing 3 polyadenylation16hThe problem(s) with RNA:RNA is chemic

12、ally unstable - spontaneous cleavage of phosphodiester backbone via intramolecular transesterificationRNA is susceptible to nearly ubiquitous RNA-degrading enzymes (RNases)RNases are released upon cell lysisRNases are present on the skinRNases are very difficult to inactivate- disulfide bridges conf

13、erring stability- no requirement for divalent cations for activity17hCommon sources of RNase and how to avoid themContaminated solutions/buffersUSE GOOD STERILE TECHNIQUETREAT SOLUTIONS WITH DEPC (when possible)MAKE SMALL BATCHES OF SOLUTIONSContaminated equipmentUSE “RNA-ONLY” PIPETS, GLASSWARE, GE

14、L RIGSBAKE GLASSWARE, 300C, 4 hoursUSE “RNase-free” PIPET TIPSTREAT EQUIPMENT WITH DEPC18hTop 10 sources of RNase contamination(Ambion Scientific website)Ungloved handsTips and tubesWater and buffersLab surfacesEndogenous cellular RNasesRNA samplesPlasmid prepsRNA storage (slow action of small amoun

15、ts of RNAseChemical nucleases (Mg2+, Ca2+at 80C for 5 +) Enzyme preparations19hInhibitors of RNaseDEPC: diethylpyrocarbonatealkylating agent, modifying proteins and nucleic acids fill glassware with 0.1% DEPC, let stand overnight at room tempsolutions may be treated with DEPC - add DEPC to 0.1%, the

16、n autoclave (DEPC breaks down to CO2 and ethanol)20hInhibitors of RnaseVanadyl ribonucleoside complexescompetitive inhibitors of RNAses, but need to be removed from the final preparation of RNAProtein inhibitors of RNasehorseshoe-shaped, leucine rich protein, found in cytoplasm of most mammalian tis

17、suesmust be replenished following phenol extraction steps21hMaking and using mRNA (1)22hMaking and using mRNA (2)23hPurifying RNA: the key is speedBreak the cells/solubilize components/inactivate RNAses by the addition of guanidinium thiocyanate (very powerful denaturant)Extract RNA using phenol/chl

18、oroform (at low pH)Precipitate the RNA using ethanol/LiClStore RNA:in DEPC-treated H20 (-80C)in formamide (deionized) at -20C24hSelective capture of mRNA: oligo dT-celluloseOligo dT is linked to cellulose matrixRNA is washed through matrix at high salt concentrationNon-polyadenylated RNAs are washed

19、 throughpolyA RNA is removed under low-salt conditions(not all of the non-polyadenylated RNA gets removed25hOther methods to capture mRNAPoly(U) sepharose chromatographyPoly(U)-coated paper filtersStreptavidin beads: A biotinylated oligo dT is added to guanidinium-treated cells, and it anneals to th

20、e polyA tail of mRNAsBiotin/streptavidin interactions permit isolation of the mRNA/oligo dT complexes26hHow good is the RNA prep?The rRNA should form 2 sharp bands in ethidium bromide-stained gels (but mRNA will not be visibleUse radiolabelled poly dT in a pilot Northern hybridization-should get a s

21、mear from 0.6 to 5 kb on the blotUse a known, “standard” gene probe (e.g. GAPDH in mammalian cells) in Northern hybridization-there should be a sharp band with no degradation products27hVisualizing DNA and RNA: non-specific methodsQuantitation of DNAElectrophoresisVisualizing DNA in gels 28hVisualiz

22、ing DNA: ElectrophoresisAllows separation of biomolecules (DNA, RNA, protein) on basis of sizeA separation matrix, or gel (agarose or polyacrylamide), is saturated with an electrically conductive bufferSamples are loaded, an electric field is applied, and negatively charged biomolecules in the sampl

23、e travel toward the cathode The larger the molecule, the slower the travel through the gel matrixDyes allow a visual estimate of the rate of travel through the gelThe choice of matrix depends mainly on the size of DNA being analyzed29hAgarose gelsAgarose: a polysaccharide polymer of alternating D- a

24、nd L-galactose monomers, isolated from seaweedPore size is defined by the agarose concentration (higher concentration, slower DNA migration overall)The conformation of the DNA (supercoiled, nicked circles, linear) affects the mobility of the DNA in gels Rate of DNA migration is affected by voltage (

25、5 to 8 Volts/cm is close to optimal)Agarose comes in a myriad of types (variable melting temperatures, generated by differential hydroxyethylation of the agarose)30hAgarose gelsStandard gels can separate DNA fragments from 100 bp to about 20,000 bpPulsed-field gels separate very large DNA fragments

26、(up to 10,000,000 bp, or 10 Mb)This apparatus allows periodic shifts in the direction of DNA migration: 120 refers to the reorientation angle (difference between orientation of electric fields A and B31h-+time of electrophoresis(progress monitored by marker dyes)Load samples in wells bromophenolBlue

27、(500bp)xyleneCyanol(4000bp)Typical agarose gel(the DNA fragments are not visible without some sort of staining) 32h33hPolyacrylamide gelsAcrylamide monomers (toxic!) polymerized to form gel matrixThe gel structure is held together by the cross-linker- usually N, N-methylenebisacrylamide (bis for sho

28、rt)Pore size defined by concentration of gel (total percentage) and concentration of the crosslinker (bis) relative to acrylamide monomerVery high resolution (better than agarose)Suitable for separation of nucleic acids from 6 to 1000 base pairs in length34hPolyacrylamide gelsNative gels (DNA stays

29、double-stranded)Denaturing gels-run in the presence of high concentrations of denaturant (usually urea) and at high temperature: DNA is single stranded (sequencing gels)(also useful in separation of proteins, when proteins are treated with SDS, which denatures proteins and gives a uniformly negative

30、 surface charge)35hRecipe for a polyacrylamide gel:Acrylamide (anywhere from 4 to 20 %, depending size of nucleic acids or proteins in the gel)Bis-acrylamide (the ratio of Bis to regular acrylamide is important)WaterBufferTo initiate polymerization, addAPS: Ammonium persulfate - generates free radic

31、als needed for polymerizationTEMED: N,N,N,N - tetramethylethylenediamine - accelerates free radical generation by APS36hMore about gelsThere has to be a buffer (for carrying current)TAE (Tris-acetate-EDTA): good resolution of DNA, but buffering capacity is quickly depletedTBE (Tris-borate-EDTA): Hig

32、h buffering capacity, resolution is pretty goodUse gel loading “buffers” (relatively simple)Dense material to carry sample to bottom of wells (sucrose, glycerol, or ficoll)Dyes for tracking progress of electrophoresisBromophenol blue: fast migrationXylene cyanol: slow migrationOccasionally denaturan

33、t is present (formamide) for denaturing gels (e.g. sequencing gels)37hethidium bromide, an anti-trypanosomal drug for cattleStain works by intercalating in stacked base pairs, elongates DNA helixFluorescence increases upon DNA bindingStained bands visualized by UV illumination (302 or 260 nm)Stainin

34、g nucleic acidsEthidium bromideG-C base pair38hExample of an agarose-DNA gel, Stained with ethidium bromide Direction ofelectrophoresisFragments of bacteriophage genomic DNA (48 kb) cut with the restriction enzyme Hind IIIThe fragments are equimolar-why is the band intensity different?39hAnother eth

35、idium bromide-stained agarose gelThe marker lane (M) gives size standards for comparison with the sample lanesMsamples40hOther methods for staining DNA SYBR gold (Molecular Probes, Eugene, OR), more than 10-fold more sensitive than ethidium bromide for detecting DNA, but expensive! methylene blue: n

36、ot toxic, but the staining protocol is time consuming, and sensitivity somewhat lower than ethidium bromide silver staining: high degree of sensitivity, but the protocols are time consuming, and proteins are also stained by silver41hSouthern blots (DNA-DNA hybridizationB. Northern blots (DNA-RNA hyb

37、ridization)MicroarrayMethods for detecting specific DNA/RNA42hVisualizing DNA, RNA and Protein: detecting specific sequencesTechniques allow one to distinguish specific sequences or proteins in a large, mixed population, e.g. in cell extracts or genomic DNA preparationsFor DNA and RNA, specific sequ

38、ence detection is based on DNA and RNA complementarity and base-pairing 43hDetecting specific DNA sequences: the Southern blot44hAgarose orPolyacrylamide gelnitrocelluloseor nylon membrane boundary:DNA binds to itA typical capillary blotting apparatus. Electroblotting is also commonly usedImmobiliza

39、tion of nucleic acids45hDNA is fixed to the nylon membrane by:Baking, 80CUV crosslinking (links thymines in DNA to + charged amine groups in membrane), DNA onlyProbe to detect sequence of interest by base-pairing (hybridization)Obtain probe DNA: synthetic oligonucleotide or cloned gene (single stran

40、ded)Label probe for later detectionRadioactivityNon-radioactive labelSouthern blotting:Immobilization of target DNA and detection46h Use T4 polynucleotide kinase-catalyzes the transfer of the gamma phosphate of 32P ATP to the 5 end of DNA fragment to be used as a probe 32P is a high energy beta part

41、icle emitter, and provides good sensitivity for detection of hybridization between the probe DNA and the target (blot) DNA Detect radiolabel with -autoradiography (X ray film)-phosphorimager (phosphor coated plates store the energy of the radioactive particle, laser excitation releases photons of li

42、ght that are collected and represented as a picture, greater dynamic range than film, and faster tooRadioactive probes: 32P labeling47he.g. horseradish peroxidaseoxidationNon-radioactive labels48hor digoxygenin/antibody-conjugated HRPcan also usebiotinylated DNA probeoxidationNon-radioactive labels4

43、9hblocking agents (e.g. milk, SDS) prevent non-specific interactions between probes and membraneVolume exclusion agents (eg. dextran sulfate) increase rate and level of hybridizationWash blot with increasing stringencyLow stringency: high salt, low temperature, probe binds to sequences with mismatch

44、esHigh stringency: low salt, higher temp., probe binds only to fully complementary sequencesHybridize probes to membranes50hSouthern Blot-one example(RFLPs)(or PCR fragment)51hSame basic technique as Southern blots, but RNA is run on the initial gel and is transferred to the membrane.Use this method

45、 to measure levels of gene transcription in vivo (detecting changes in the levels of RNA transcript under differing conditions)Microarrays for measuring mRNA abundance are based on this principle, but many probes are immobilized in a regular array - reverse transcribed (and fluorescently labelled) R

46、NA “lights up” the probes on the microarrayNorthern blots:52hQuantitation of DNA by UV absorbance Measure absorbance of UV light by sample (the aromatic bases have a characteristic absorbance maximum at around 260 nanometers) 1.0 A260 (1 cm light path) = DNA concentration of 50 micrograms per ml (double stranded DNA) or 38 micrograms per ml (single-stranded DNA or RNA) the effective range for accurate measurement is rather narrow: A260 from 0.05 to 2.0 (DNA concentrations from 2.5 to 100 micro