杨荣武分子生物学课件Week2modification.ppt

DNA and RNA isolation, purification, visualization and quantitation,Genomic DNA preparation overview Plasmid DNA preparation DNA purification Phenol extraction Ethanol precipitation RNA work,What do we need DNA for?,Detect, enumerate, clone genes Detect, enumerate species Detect/sequence specific DNA regions Create 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?,cell growth,cell harvest and lysis,DNA purification,DNA purification: overview,DNA concentration,Bacterial genomic DNA prep: cell extract,Lysis: Detergents Organic solvent Proteases (lysozyme) Heat,“cell extract”,Genomic DNA prep: removing proteins and RNA,Add the enzyme RNase to degrade RNA in the aqueous layer,Need to mix gently! (to avoid shearing breakage of the genomic DNA),chloroform,2 ways to concentrate the genomic DNA,70% final conc.,“spooling”,Ethanol precipitation,Genomic DNA prep in plants - how get rid of carbohydrates?,CTAB: Cationic detergent (MC 6.61-6.62),(low ionic conditions),N+,CH3,Br-,CH3,CH3,C16H33,Plasmids: vehicles of recombinant DNA,Bacterial cell,genomic DNA,plasmids,Non-chromosomal DNA Replication: independent of the chromosome Many copies per cell Easy to isolate Easy to manipulate,Plasmid purification: alkaline lysis,Alkaline conditions denature DNA Neutralize: genomic DNA cant renature (plasmids CAN because they never fully separate),DNA purification: silica binding,Binding occurs in presence of high salt concentration, and is disrupted by elution with water,DNA purification: phenol/chloroform extraction 1:1 phenol : chloroform or 25:24:1 phenol : chloroform : isoamyl alcohol Phenol: denatures proteins, precipitates form at interface between aqueous and organic layer Chloroform: increases density of organic layer Isoamyl alcohol: prevents foaming,Aqueous volume (at least 200 microliters) Add 2 volumes of phenol:chloroform, mix well Spin in centrifuge, move aqueous phase to a new tube Repeat steps 2 and 3 until there is no precipitate at phase interface (extract aqueous layer with 2 volumes of chloroform),Phenol extraction,Ethanol depletes the hydration shell surrounding DNA Allowing cations to interact with the DNA phosphates Reducing repulsive forces between DNA strands Causing aggregation and precipitation of DNA Aqueous volume (example: 200 microliters) - add 22 microliters sodium acetate 3M pH 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 concentration),cell growth,cell harvest and lysis,DNA purification,DNA purification: overview,DNA concentration,Isolation of RNA - Course reading 11,DNA - mRNA - protein,Lots 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 mRNA measurement really say about expression of the protein?),RNA in a typical eukaryotic cell: 10-5 micrograms RNA 80-85% is ribosomal RNA 15-20% is small RNA (tRNA, small nuclear RNAs) About 1-5% is mRNA - variable in size - but usually containing 3 polyadenylation,The problem(s) with RNA: RNA is chemically unstable - spontaneous cleavage of phosphodiester backbone via intramolecular transesterification RNA is susceptible to nearly ubiquitous RNA-degrading enzymes (RNases) RNases are released upon cell lysis RNases are present on the skin RNases are very difficult to inactivate - disulfide bridges conferring stability - no requirement for divalent cations for activity,Common sources of RNase and how to avoid them,Contaminated solutions/buffers USE GOOD STERILE TECHNIQUE TREAT SOLUTIONS WITH DEPC (when possible) MAKE SMALL BATCHES OF SOLUTIONS Contaminated equipment USE “RNA-ONLY” PIPETS, GLASSWARE, GEL RIGS BAKE GLASSWARE, 300C, 4 hours USE “RNase-free” PIPET TIPS TREAT EQUIPMENT WITH DEPC,Top 10 sources of RNase contamination (Ambion Scientific website) Ungloved hands Tips and tubes Water and buffers Lab surfaces Endogenous cellular RNases RNA samples Plasmid preps RNA storage (slow action of small amounts of RNAse Chemical nucleases (Mg2+, Ca2+at 80C for 5 +) Enzyme preparations,Inhibitors of RNase DEPC: diethylpyrocarbonate alkylating agent, modifying proteins and nucleic acids fill glassware with 0.1% DEPC, let stand overnight at room temp solutions may be treated with DEPC - add DEPC to 0.1%, then autoclave (DEPC breaks down to CO2 and ethanol),Inhibitors of Rnase Vanadyl ribonucleoside complexes competitive inhibitors of RNAses, but need to be removed from the final preparation of RNA Protein inhibitors of RNase horseshoe-shaped, leucine rich protein, found in cytoplasm of most mammalian tissues must be replenished following phenol extraction steps,Making and using mRNA (1),Making and using mRNA (2),Purifying RNA: the key is speed Break the cells/solubilize components/inactivate RNAses by the addition of guanidinium thiocyanate (very powerful denaturant) Extract RNA using phenol/chloroform (at low pH) Precipitate the RNA using ethanol/LiCl Store RNA: in DEPC-treated H20 (-80C) in formamide (deionized) at -20C,Selective capture of mRNA: oligo dT-cellulose Oligo dT is linked to cellulose matrix RNA is washed through matrix at high salt concentration Non-polyadenylated RNAs are washed through polyA RNA is removed under low-salt conditions (not all of the non-polyadenylated RNA gets removed,Other methods to capture mRNA Poly(U) sepharose chromatography Poly(U)-coated paper filters Streptavidin beads: A biotinylated oligo dT is added to guanidinium-treated cells, and it anneals to the polyA tail of mRNAs Biotin/streptavidin interactions permit isolation of the mRNA/oligo dT complexes,How good is the RNA prep? The rRNA should form 2 sharp bands in ethidium bromide-stained gels (but mRNA will not be visible Use radiolabelled poly dT in a pilot Northern hybridization-should get a smear from 0.6 to 5 kb on the blot Use a known, “standard” gene probe (e.g. GAPDH in mammalian cells) in Northern hybridization-there should be a sharp band with no degradation products,Visualizing DNA and RNA: non-specific methods,Quantitation of DNA Electrophoresis Visualizing DNA in gels,Visualizing DNA: Electrophoresis,Allows separation of biomolecules (DNA, RNA, protein) on basis of size A separation matrix, or gel (agarose or polyacrylamide), is saturated with an electrically conductive buffer Samples are loaded, an electric field is applied, and negatively charged biomolecules in the sample travel toward the cathode The larger the molecule, the slower the travel through the gel matrix Dyes allow a visual estimate of the rate of travel through the gel The choice of matrix depends mainly on the size of DNA being analyzed,Agarose gels,Agarose: a polysaccharide polymer of alternating D- and L-galactose monomers, isolated from seaweed Pore 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 (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),Agarose gels,Standard gels can separate DNA fragments from 100 bp to about 20,000 bp Pulsed-field gels separate very large DNA fragments (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 B,-,+,time of electrophoresis (progress monitored by marker dyes),Load samples in wells,bromophenol Blue(500bp),xylene Cyanol(4000bp),Typical agarose gel,(the DNA fragments are not visible without some sort of staining),Polyacrylamide gels,Acrylamide monomers (toxic!) polymerized to form gel matrix The gel structure is held together by the cross-linker- usually N, N-methylenebisacrylamide (“bis“ for short) Pore size defined by concentration of gel (total percentage) and concentration of the crosslinker (bis) relative to acrylamide monomer Very high resolution (better than agarose) Suitable for separation of nucleic acids from 6 to 1000 base pairs in length,Polyacrylamide gels,Native gels (DNA stays 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 surface charge),Recipe 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) Water Buffer To initiate polymerization, add APS: Ammonium persulfate - generates free radicals needed for polymerization TEMED: N,N,N,N - tetramethylethylenediamine - accelerates free radical generation by APS,More about gels There has to be a buffer (for carrying current) TAE (Tris-acetate-EDTA): good resolution of DNA, but buffering capacity is quickly depleted TBE (Tris-borate-EDTA): High buffering capacity, resolution is pretty good Use gel loading “buffers” (relatively simple) Dense material to carry sample to bottom of wells (sucrose, glycerol, or ficoll) Dyes for tracking progress of electrophoresis Bromophenol blue: fast migration Xylene cyanol: slow migration Occasionally denaturant is present (formamide) for denaturing gels (e.g. sequencing gels),ethidium bromide, an anti-trypanosomal drug for cattle Stain works by intercalating in stacked base pairs, elongates DNA helix Fluorescence increases upon DNA binding Stained bands visualized by UV illumination (302 or 260 nm),Staining nucleic acids,Ethidium bromide,G-C base pair,Example of an agarose-DNA gel, Stained with ethidium bromide,Direction of electrophoresis,Fragments of bacteriophage genomic DNA (48 kb) cut with the restriction enzyme Hind III The fragments are equimolar-why is the band intensity different?,Another ethidium bromide-stained agarose gel,The marker lane (M) gives size standards for comparison with the sample lanes,M,samples,Other 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: not 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 silver,Southern blots (DNA-DNA hybridization B. Northern blots (DNA-RNA hybridization) Microarray,Methods for detecting specific DNA/RNA,Visualizing DNA, RNA and Protein: detecting specific sequences,Techniques allow one to distinguish specific sequences or proteins in a large, mixed population, e.g. in cell extracts or genomic DNA preparations For DNA and RNA, specific sequence detection is based on DNA and RNA complementarity and base-pairing,Detecting specific DNA sequences: the Southern blot,Agarose or Polyacrylamide gel,nitrocellulose or nylon membrane boundary: DNA binds to it,A typical capillary blotting apparatus. Electroblotting is also commonly used,Immobilization of nucleic acids,DNA is fixed to the nylon membrane by: Baking, 80C UV crosslinking (links thymines in DNA to + charged amine groups in membrane), DNA only Probe to detect sequence of interest by base-pairing (hybridization) Obtain probe DNA: synthetic oligonucleotide or cloned gene (single stranded) Label probe for later detection Radioactivity Non-radioactive label,Southern blotting: Immobilization of target DNA and detection,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 particle 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 light that are collected and represented as a picture, greater dynamic range than film, and faster too,Radioactive probes: 32P labeling,e.g. horseradish peroxidase,oxidation,Non-radioactive labels,or digoxygenin/antibody-conjugated HRP,can also use biotinylated DNA probe,oxidation,Non-radioactive labels,blocking agents (e.g. milk, SDS) prevent non-specific interactions between probes and membrane Volume exclusion agents (eg. dextran sulfate) increase rate and level of hybridization Wash blot with increasing stringency Low stringency: high salt, low temperature, probe binds to sequences with mismatches High stringency: low salt, higher temp., probe binds only to fully complementary sequences,Hybridize probes to membranes,Southern Blot-one example,(RFLPs),(or PCR fragment),Same basic technique as Southern blots, but RNA is run on the initial gel and is transferred to the membrane. Use this method 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) RNA “lights up” the probes on the microarray,Northern blots:,Quantitation 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 micrograms/ml) S