Bài giảng Molecular Biology - Chapter 5 Molecular Tools for Studying Genes and Gene Activity

Molecular BiologyFifth EditionChapter 5Molecular Tools for Studying Genes and Gene ActivityLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.15.1 Molecular Separations• Often mixtures of proteins or nucleic acids are generated during the course of molecular biological proceduresA protein may need to be purified from a crude cellular extractA particular nucleic acid molecule made in a reaction needs to be purified Gel electrophoresis is used to separate different species of: Nucleic acidProtein2DNA Gel ElectrophoresisMelted agarose is poured into a form equipped with removable comb Comb “teeth” form slots in the solidified agaroseDNA samples are placed in the slotsAn electric current is run through the gel at a neutral pH to allow the sample to travel through the gel matrix3DNA Separation by Agarose Gel ElectrophoresisDNA is negatively charged due to the phosphates in its backbone and moves toward the positive poleSmall DNA pieces have little frictional drag so they move rapidlyLarge DNAs have more frictional drag so their mobility is slowerDistributes DNA according to sizeLargest near the topSmallest near the bottomDNA is stained with fluorescent dye that intercalates between the bases4DNA Size EstimationMobility of fragments are plotted v. log of molecular weight (or number of base pairs)Electrophoresis of unknown DNA in parallel with standard fragments permits size estimation upon comparisonSame principles apply to RNA separation5Electrophoresis of Large DNASpecial techniques are required for DNA fragments larger than about 1 kilobasesInstead of constant current, alternate long pulses of current in forward direction with shorter pulses in either opposite or sideways directionTechnique is called pulsed-field gel electrophoresis (PFGE)6Protein Gel ElectrophoresisSeparation of proteins is done using polyacrylamide gel electrophoresis (PAGE)Treat proteins to denature subunits with detergent such as sodium dodecyl sulfate (SDS)SDS coats polypeptides with negative charges so all move to anodeMasks natural charges of protein subunits so all move relative to mass not chargeAs with DNA smaller proteins move faster toward the anode7SummaryDNAs, RNAs, and proteins of various masses can be separated by gel electrophoresisMost common gel used in nucleic acid electrophoresis is agarose but polyacrylamide is typically used in protein electrophoresisSDS-PAGE is used to separate polypeptides according to their masses8Two-Dimensional Gel ElectrophoresisWhile SDS-PAGE gives good resolution of polypeptides, some mixtures are so complex that additional resolution is neededTwo-dimensional gel electrophoresis:Nondenaturing gel electrophoresis (no SDS) uses 2 consecutive gels each in a different dimensionSequential gels with distinct pH separation and polyacrylamide gel concentration9A Simple 2-D MethodSamples are run in 2 gelsFirst dimension separates using one concentration of polyacrylamide at one pHSecond dimension uses different concentration of polyacrylamide and pHProteins move differently at different pH values without SDS and at different acrylamide concentrations 10A More Powerful Two-Dimensional Gel Electrophoresis TechniqueA two process method:Isoelectric focusing gel: mixture of proteins electrophoresed through gel in a narrow tube containing a pH gradientNegatively charged protein moves to its isoelectric point at which it is no longer chargedTube gel is removed and used as the sample in the second process11Standard SDS-PAGE:Tube gel is removed and used as the sample at the top of a standard polyacrylamide gelProteins partially resolved by isoelectric focusing are further resolved according to sizeWhen used to a compare complex mixtures of proteins prepared under two different conditions, even subtle differences are visibleA More Powerful Two-Dimensional Gel Electrophoresis Technique continued12Ion-Exchange ChromatographyChromatography originally referred to the pattern seen after separating colored substances on paperIon-exchange chromatography uses a resin to separate substances by chargeThis is especially useful for proteinsResin is placed in a column and the sample is loaded onto the column material13Separation by Ion-Exchange ChromatographyOnce the sample is loaded buffer is passed over the resin + sampleAs ionic strength of elution buffer increases, samples of solution flowing through the column are collectedSamples are tested for the presence of the protein of interest14Gel Filtration ChromatographyProtein size is a valuable property that can be used as a basis of physical separation Gel filtration uses columns filled with porous resins that let in smaller substances and exclude larger substancesAs a result larger substances travel faster through the column15Affinity ChromatographyThe resin contains a substance to which the molecule of interest has a strong and specific affinityThe molecule binds to a column resin coupled to the affinity reagentMolecule of interest is retainedMost other molecules flow through without bindingLast, the molecule of interest is eluted from the column using a specific solution that disrupts their specific binding16SummaryHigh-resolution separation of proteins can be achieved by two-dimensional gel electrophoresisIon-exchange chromatography can be used to separate substances according to their sizesGel filtration chromatography uses columns filled with porous resins that let in smaller substances but exclude larger onesAffinity chromatography is a powerful purification technique that exploits an affinity reagent with strong and specific affinity for a molecule of interest175.2 Labeled TracersFor many years “labeled” has been synonymous with “radioactive”Radioactive tracers allow vanishingly small quantities of substances to be detectedMolecular biology experiments typically require detection of extremely small amounts of a particular substance18AutoradiographyAutoradiography is a means of detecting radioactive compounds with a photographic emulsionPreferred emulsion is x-ray filmDNA is separated on a gel and radiolabeledGel is placed in contact with x-ray film for hours or daysRadioactive emissions from the labeled DNA expose the filmDeveloped film shows dark bands19Autoradiography AnalysisRelative quantity of radioactivity can be assessed looking at the developed filmMore precise measurements are made using a densitometerArea under peaks on a tracing by a scanner Proportional to darkness of the bands on autoradiogram20PhosphorimagingThis technique is more accurate in quantifying amount of radioactivity in a substanceResponse to radioactivity is much more linearPlace gel with radioactive bands in contact with a phosphorimager platePlate absorbs b electrons that excite molecules on the plate which remain excited until plate is scannedMolecular excitation is monitored by a detector21Liquid Scintillation CountingRadioactive emissions from a sample create photons of visible light are detected by a photomultiplier tube in the process of liquid scintillation countingRemove the radioactive material (band from gel) to a vial containing scintillation fluidFluid contains a fluor that fluoresces when hit with radioactive emissionsActs to convert invisible radioactivity into visible light22Nonradioactive TracersNewer nonradioactive tracers now rival older radioactive tracers in sensitivityThese tracers do not have hazards:Health exposureHandlingDisposalIncreased sensitivity is from use of a multiplier effect of an enzyme that is coupled to probe for molecule of interest23Detecting Nucleic Acids With a Nonradioactive Probe245.3 Using Nucleic Acid HybridizationHybridization is the ability of one single-stranded nucleic acid to form a double helix with another single strand of complementary base sequencePrevious discussion focused on colony and plaque hybridizationThis section looks at techniques for isolated nucleic acids25Southern Blots: Identifying Specific DNA FragmentsDigests of genomic DNA are separated on a gelThe separated pieces are transferred to filter (nitrocellulose) by diffusion, or more recently by electrophoresing the DNA onto the filterThe filter is then treated with alkali to denature the DNA, resulting ssDNA binds to the filterA labeled cDNA probe that is complementary to the DNA of interest is then applied to the filterA positive band should be detectable where hybridization between the probe and DNA occurred26Southern BlotsThe probe hybridizes and a band is generated corresponding to the DNA fragment of interestVisualize bands with x-ray film or autoradiographyMultiple bands can lead to several interpretationsMultiple genesSeveral restriction sites in the gene27DNA Fingerprinting and DNA TypingSouthern blots are used in forensic labs to identify individuals from DNA-containing materialsMinisatellite DNA is a sequence of bases repeated several times, also called a DNA fingerprintIndividuals differ in the pattern of repeats of the basic sequenceThe difference is large enough that 2 people have only a remote chance of having exactly the same pattern of repeats28DNA FingerprintingProcess is a Southern blot Cut the DNA under study with restriction enzymeIdeally cut on either side of minisatellite but not inside Run the digested DNA on a gel and blot Probe with labeled minisatellite DNA and imageNote that real samples result in very complex patterns29Forensic Uses of DNA Fingerprinting and DNA TypingWhile people have different DNA fingerprints, parts of the pattern are inherited in a Mendelian fashionCan be used to establish parentagePotential to identify criminalsRemove innocent people from suspicion Actual pattern has so many bands they can smear together indistinguishablyForensics uses probes for just a single locusSet of probes gives a set of simple patterns30In Situ Hybridization: Locating Genes in ChromosomesLabeled probes can be used to hybridize to chromosomes and reveal which chromosome contains the gene of interestSpread chromosomes from a cellPartially denature DNA creating single-stranded regions to hybridize to labeled probeStain chromosomes and detect presence of label on particular chromosomeProbe can be detected with a fluorescent antibody in a technique called fluorescence in situ hybridization (FISH)31ImmunoblotsImmunoblots (also called Western blots) use a similar process to Southern blotsElectrophoresis of proteinsBlot the proteins from the gel to a membraneDetect the protein using antibody or antiserum to the target proteinLabeled secondary antibody is used to bind the first antibody for visualization and to increase the signal32SummaryLabeled DNA (or RNA) probes can be used to hybridize to DNAs of the same or very similar sequence on a Southern blotDNA fingerprinting can be used as a forensic tool or to test parentageIn situ hybridization can be used to locate genes or other specific DNA sequences on whole chromosomesProteins can be detected and quantified in a complex mixture using Western blots335.4 DNA SequencingSanger, Maxam, and Gilbert developed 2 methods for determining the exact base sequence of a cloned piece of DNAModern DNA sequencing is based on the Sanger method and uses dideoxy nucleotides to terminate DNA synthesisThe process yields a series of DNA fragments whose size is measured by electrophoresisThe last base in each fragment is known as that dideoxy nucleotide was used to terminate the reactionOrdering the fragments by size tells the base sequence of the DNA34Sanger Method of DNA Sequencing35Automated DNA SequencingManual sequencing is powerful but slowAutomated sequencing uses dideoxynucleotides tagged with different fluorescent moleculesProducts of each dideoxynucleotide will fluoresce a different colorFour reactions are completed, then mixed together and run out on one lane of a gel36High Throughput SequencingOnce an organism’s genome sequence is known, very rapid sequencing techniques can be applied to sequence the genome of another member of the same speciesProduces relatively short reads or contiguous sequences (25-35bp or 200-300bp, depending on the method) that can easily be pieced together if a reference sequence is available37High Throughput SequencingPyrosequencing is one example that is an automated system with the advantages of speed and accuracy - nucleotides are added one by one and the incorporation of a nucleotide is detected by a release of pyrophosphate, which leads to a flash of lightAnother method (Illumina company) starts by attaching short pieces of DNA to a solid surface, amplifying each DNA in a tiny patch on the surface, then sequencing the patches together by extending them one nucleotide at a time using fluorescent chain-terminating nucleotides, whose fluoresce reveals their identity38Restriction MappingPrior to the start of large-scale sequencing preliminary work is done to locate landmarksA map based on physical characteristics is called a physical mapIf restriction sites are the only map features then a restriction map has been prepared39Restriction Map ExampleConsider a 1.6 kb piece of DNA as an exampleCut separate samples of the original 1.6 kb fragment with different restriction enzymesSeparate the digests on an agarose gel to determine the size of pieces from each digestCan also use same digest to find the orientation of an insert cloned into a vector40Southern Blots and Restriction Mapping41SummaryPhysical maps tell about the spatial arrangement of physical “landmarks” such as restriction sitesIn restriction mapping cut the DNA in question with 2 or more restriction enzymes in separate reactionsMeasure the sizes of the resulting fragmentsCut each with another restriction enzyme and measure size of subfragments by gel electrophoresisSizes permit location of some restriction sites relative to othersImprove process by Southern blotting fragments and hybridizing them to labeled fragments from another restriction enzyme to reveal overlaps425.5 Protein Engineering With Cloned Genes: Site-Directed MutagenesisCloned genes permit biochemical microsurgery on proteinsSpecific bases in a gene may be changedAmino acids at specific sites in the protein product may be altered as a resultEffects of those changes on protein function can be observed43PCR-based Site-Directed Mutagenesis44SummaryUsing cloned genes, one can introduce changes that may alter the amino acid sequence of the corresponding protein productsMutagenized DNA can be made with:Double-stranded DNATwo complementary mutagenic primersPCRDigest the PCR product to remove wild-type DNACells can be transformed with mutagenized DNA455.6 Mapping and Quantifying TranscriptsIn the field of molecular biology mapping (locating start and end) and quantifying (how much transcript exists at a set time) transcripts are common proceduresOften transcripts do not have a uniform terminator, resulting in a continuum of species smeared on a gelTechniques that are specific for the sequence of interest are important46Northern BlotsNorthern blots detect RNAExample: You have cloned a cDNAQuestion: How actively is the corresponding gene expressed in different tissues?Answer: Find out using a Northern BlotObtain RNA from different tissuesRun RNA on agarose gel and blot to membraneHybridize to a labeled cDNA probeNorthern plot tells abundance of the transcriptQuantify using densitometer47S1 MappingUse S1 mapping to locate the ends of RNAs and to determine the amount of a given RNA in cells at a given timeLabel a ssDNA probe that can only hybridize to transcript of interestProbe must span the sequence start to finishAfter hybridization, treat with S1 nuclease which degrades ssDNA and RNATranscript protects part of the probe from degradationSize of protected area can be measured by gel electrophoresis48S1 Mapping the 5’ End49S1 Mapping the 3’ End50SummaryA Northern blot is similar to a Southern blot but is a method used for detection of RNA In S1 mapping, a labeled DNA probe is used to detect 5’- or 3’-end of a transcriptAmount of probe protected is proportional to concentration of transcript, so S1 mapping can be quantitativeRNase mapping is a variation on SI mapping that uses an RNA probe and RNase51Primer Extension SchematicStart with in vivo transcription, harvest cellular RNA containing desired transcriptHybridize labeled oligonucleotide [18nt] (primer)Reverse transcriptase extends the primer to the 5’-end of transcriptDenature the RNA-DNA hybrid and run the mix on a high-resolution DNA gelCan estimate transcript concentration alsoPrimer extension works to determine the 5’-end of a transcript to one-nucleotide accuracy52Run-Off TranscriptionA good assay to measure the rate of in vitro transcriptionDNA fragment containing gene to transcribe is cut with restriction enzyme in middle of transcription regionTranscribe the truncated fragment in vitro using labeled nucleotides, as polymerase reaches truncation it “runs off” the endMeasure length of run-off transcript compared to location of restriction site at 3’-end of truncated gene53Schematic of the G-Less Cassette AssayTranscribe altered template in vitro with CTP, ATP and UTP one of which is labeled, but no GTPTranscription will stop when the first G is required resulting in an aborted transcript of predictable sizeSeparate transcripts on a gel and measure transcription activity with autoradiographyA variation of the run-off technique in which a stretch of nucleotides lacking guanines is inserted into the nontemplate strand just downstream of the promoter54SummaryRun-off transcription is a means of checking efficiency and accuracy of in vitro transcriptionGene is truncated in the middle and transcribed in vitro in presence of labeled nucleotidesRNA polymerase runs off the end making an incomplete transcriptSize of run-off transcript locates transcription start siteAmount of transcript reflects efficiency of transcriptionIn G-less cassette transcription, a promoter is fused to dsDNA cassette lacking Gs in nontemplate strandConstruct is transcribed in vitro in absence of of GTPTranscription aborts at end of cassette for a predictable size band on a gel555.7 Measuring Transcription Rates in VivoPrimer extension, S1 mapping and Northern blotting will determine the concentration of specific transcripts at a given timeThese techniques do not really reveal the rate of transcript synthesis as concentration involves both:Transcript synthesisTranscript degradation56Nuclear Run-On TranscriptionThe idea of this assay is to isolate nuclei from cells, allow them to extend in vitro the transcripts already started in vivo RNA polymerase that has already initiated transcription will “run-on” or continue to elongate the same RNA chainsEffective as initiation of new RNA chains in isolated nuclei does not generally occurResults will show transcription rates and an idea of which genes are transcribed57Nuclear Run-On Transcription Diagram58Reporter Gene TranscriptionPlace a surrogate reporter gene under the control of a specific promoter and measure the accumulation of the product of this reporter geneThe reporter genes are carefully chosen to have products very convenient to assaylacZ produces b-galactosidase which has a blue cleavage productcat produces chloramphenicol acetyl transferase (CAT) which inhibits bacterial growthLuciferase produces a chemiluminescent compound that emits light59Measuring Protein Accumulation in VivoGene activity can be monitored by measuring the accumulation of protein, the ultimate gene productThere are two primary methods of measuring protein accumulationImmunoblotting / Western blotting (discussed earlier)ImmunoprecipitationImmunoprecipitation typically uses an antibody that will bind specifically to the protein of interest followed with a secondary antibody complexed to Protein A on resin beads using a low-speed centrifuge605.8 Assaying DNA-Protein InteractionsStudy of DNA-protein interactions is of significant interest to molecular biologistsTypes of interactions often studied:Protein-DNA bindingWhich bases of DNA interact with a protein61Filter BindingFilter binding is used to measure DNA-protein interaction and based on the fact that double-stranded DNA will not bind by itself to a filter, but a protein-DNA complex willDouble-stranded DNA can be labeled and mixed with proteinAssay protein-DNA binding by measuring the amount of label retained on the filter62Nitrocellulose Filter-Binding AssaydsDNA is labeled and mixed with proteinPour dsDNA through a nitrocellulose filterMeasure amount of radioactivity that passed through filter and retained on filter63Gel Mobility ShiftDNA moves through a gel faster when it is not bound to proteinGel shift assays detect interaction between protein and DNA by reduction of the electrophoretic mobility of a small DNA bound to a protein64FootprintingFootprinting detects protein-DNA interaction and will show where a target lies on DNA and which bases are involved in protein bindingThree methods are very popular:DNase footprintingDimethylsulfate footprintingHydroxyl radical footprinting65DNase FootprintingProtein binding to DNA covers the binding site and protects from attack by DNase End label DNA, 1 strand only Protein binds DNA Treat complex with DNase I mild conditions for average of 1 cut per molecule Remove protein from DNA, separate strands and run on a high-resolution polyacrylamide gel66DMS FootprintingStarts the same way as DNase footprinting but then methylate with DMS at conditions for 1 methylation per DNA moleculeThe protein is then dislodged and treated to remove the methylated purines resulting in apurinic sites which breaks the DNAThe DNA fragments are then electrophoresed and autoradiograped for detection67SummaryFootprinting finds target DNA sequence or binding site of a DNA-binding proteinDNase footprinting binds protein to end-labeled DNA target, then attacks DNA-protein complex with DNase DNA fragments are electrophoresed with protein binding site appearing as a gap in the pattern where protein protected DNA from degradationDMS, DNA methylating agent is used to attack the DNA-protein complexHydroxyl radicals – copper- or iron-containing organometallic complexes generate hydroxyl radicals that break the DNA strands68Chromatin Immunoprecipitation (ChIP)ChIP is a method used to discover whether a given protein is bound to a given gene in chromatin - the DNA-protein complex that is the natural state of the DNAnin a living cellChIP uses an antibody to precipitate a particular protein in complex with DNA, and PCR to determine whether the protein binds near a particular gene69Chromatin Immunoprecipitation (ChIP)705.9 Assaying Protein-Protein InteractionsImmunoprecipitation uses an antibody that will bind specifically to the protein of interest and, using a low-speed centrifuge, will ‘pull-down’ any proteins associated with the protein of interestThe yeast-two-hybrid assay is used to demonstrate binding (even transient) between two proteinsThe yeast-two-hybrid assay can also be used to fish for unknown proteins that interact with a known protein 71The Yeast-Two Hybrid Assay725.10 Finding RNA Sequences That Interact With Other MoleculesSELEX is systematic evolution of ligands by exponential enrichmentSELEX is a method to find RNA sequences that interact with other molecules, even proteinsRNAs that interact with a target molecule are selected by affinity chromatographyConvert to dsDNA and amplify by PCRRNAs are now highly enriched for sequences that bind to the target molecule73Functional SELEXFunctional SELEX is a variation where the desired function alters RNA so it can be amplifiedIf desired function is enzymatic, mutagenesis can be introduced into the amplification step to produce variants with higher activity745.11 Knockouts and TransgenesProbing structures and activities of genes does not answer questions about the role of the gene in the life of the organismTargeted disruption of genes is now possible in several organismsWhen genes are disrupted in mice the products are called knockout miceForeign genes, called transgenes, can also be added to an organism, such as a mouse, to create transgenic mice75Stage 1 of the Knockout MouseCloned DNA containing the mouse gene to be knocked out is interrupted with another gene that confers resistance to neomycinA thymidine kinase gene is placed outside the target geneMix engineered mouse DNA with stem cells so interrupted gene will find way into nucleus and homologous recombination will occur between the altered gene and the resident, intact geneThese events are rare, many cells will need to be screened using the introduced genes76Making a Knockout Mouse: Stage 177Stage 2 of the Knockout MouseIntroduce the interrupted gene into a whole mouseInject engineered cells into a mouse blastocystImplant the embryo into a surrogate mother who will give birth to chimeric mouseTrue heterozygote results when chimera mates with a black mouse to produce brown mice, half of which will have interrupted gene78Making a Knockout Mouse: Stage 279Knockout ResultsPhenotype may not be obvious in the progeny, but still instructiveOther cases can be lethal with the mice dying before birthIntermediate effects are also common and may require monitoring during the life of the mouse80Methods to Generate Transgenic MiceTwo methods to generate transgenic mice:1. Injection of cloned foreign gene into the sperm pronucleus just after fertilization of a mouse egg but before the sperm and egg nuclei have fused to allow for insertion of the foreign DNA into the embryonic cell DNA2. Injection of cloned foreign DNA into mosue embryonic stem cells, creating transgenic ES cellsBoth methods produce chimeric mice that must undergo several rounds of breeding and selection to find true transgenic animals81SummaryTo probe the role of a gene, molecular biologists can perform targeted disruption of the corresponding gene in a mouse and then look for the effects of the mutation in the ‘knockout mouse’ or insert the foreign gene as a transgene in the ‘transgenic mouse’ 82