Molecular biology - Chapter 5: Molecular tools for studying genes and gene activity

Phenotype may not be obvious in the progeny, but still instructive Other cases can be lethal with the mice dying before birth Intermediate effects are also common and may require monitoring during the life of the mouse

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Molecular Biology Fourth 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 SeparationsOften 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 purified2Gel ElectrophoresisGel electrophoresis is used to separate different species of: Nucleic acidProtein3DNA 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 pH4DNA Separation by Agarose Gel ElectrophoresisDNA is negatively charged due to phosphates in its backbone and moves to anode, the positive poleSmall DNA pieces have little frictional drag so move rapidlyLarge DNAs have more frictional drag so their mobility is slowerResult distributes DNA according to sizeLargest near the topSmallest near the bottomDNA is stained with fluorescent dye5DNA Size EstimationComparison with standards permits 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 estimationSame principles apply to RNA separation6Electrophoresis 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)7Protein Gel ElectrophoresisSeparation of proteins is done using a gel made of polyacrylamide (polyacrylamide gel electrophoresis = PAGE)Treat proteins to denature subunits with detergent such as SDSSDS 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 anode8SummaryDNAs, RNAs, and proteins of various masses can be separated by gel electrophoresisMost common gel used in nucleic acid electrophoresis is agarosePolyacrylamide is usually used in protein electrophoresisSDS-PAGE is used to separate polypeptides according to their masses9Two-Dimensional Gel ElectrophoresisWhile SDS-PAGE gives good resolution of polypeptides, some mixtures are so complex that additional resolution is neededTwo-dimensional gel electrophoresis can be done:(no SDS) uses 2 consecutive gelsSequential gels with first a pH separation, then separate in a polyacrylamide gel10A Simple 2-D MethodRun samples 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 11Two-Dimensional Gel Electrophoresis DetailsA 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 process12More Two-Dimensional Gel Electrophoresis DetailsStandard 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 visible13Ion-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 sample loaded onto the column material14Separation 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 interest15Gel 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, exclude larger onesLarger substances travel faster through the column16Affinity ChromatographyIn affinity chromatography, the 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 the specific binding175.2 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 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 agarose gelThe separated pieces are transferred to filter by diffusion, or more recently by electrophoresing the bands onto the filterFilter is treated with alkali to denature the DNA, resulting ssDNA binds to the filterProbe the filter using labeled cDNA26Southern BlotsProbe cDNA 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 DNA fingerprintIndividuals differ in the pattern of repeats of the basic sequenceDifference is large enough that 2 people have only a remote chance of having exactly the same pattern28DNA FingerprintingProcess really just a Southern blotCut the DNA under study with restriction enzymeIdeally cut on either side of minisatellite but not insideRun digest on a gel and blotProbe with labeled minisatellite DNA and imagedReal 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 and increase the signal32Western Blots33DNA SequencingSanger, Maxam, Gilbert developed 2 methods for determining the exact base sequence of a cloned piece of DNAModern DNA sequencing is based on the Sanger method34Sanger Manual SequencingSanger DNA sequencing method uses dideoxy nucleotides to terminate DNA synthesisThe process yields a series of DNA fragments whose size is measured by electrophoresisLast 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 DNA35Sanger DNA Sequencing36Automated 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 gel37Automated DNA Sequencing38Restriction MappingPrior to 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 preparedConsider a 1.6 kb piece of DNA as an example39Restriction Map 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 vector40Mapping Experiment41Using Restriction Mapping With an Unknown DNA Sample42Mapping the Unknown43Southern Blots and Restriction Mapping44SummaryPhysical map tells 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 overlaps45Protein 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 also be alteredEffects of those changes on protein function can be observedMight investigate the role of phenolic group on tyrosine compared to phenylalanine46Site-Directed Mutagenesis With PCR47SummaryUsing cloned genes, can introduce changes at will to alter amino acid sequence of 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 DNA485.4 Mapping and Quantifying TranscriptsMapping (locating start and end) and quantifying (how much transcript exists at a set time) are common proceduresOften transcripts do not have a uniform terminator, resulting in a continuum of species smeared on a gelTechniques that specific for the sequence of interest are important49Northern BlotsYou have cloned a cDNAHow actively is the corresponding gene expressed in different tissues?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 densitometer50S1 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 electrophoresis51S1 Mapping the 5’ End52S1 Mapping the 3’ End53SummaryIn S1 mapping, a labeled DNA probe is used to detect 5’- or 3’-end of a transcriptHybridization of the probe to the transcript protects a portion of the probe from digestion by S1 nuclease, specific for single-stranded polynucleotidesLength of the section of probe protected by the transcript locates the end of the transcript relative to the known location of an end of the probeAmount of probe protected is proportional to concentration of transcript, so S1 mapping can be quantitativeRNase mapping uses an RNA probe and RNase54Primer ExtensionPrimer extension works to determine exactly the 5’-end of a transcript to one-nucleotide accuracySpecificity of this method is due to complementarity between primer and transcriptS1 mapping will give similar results but limits:S1 will “nibble” ends of RNA-DNA hybridAlso can “nibble” A-T rich regions that have meltedMight not completely digest single-stranded regions55Primer 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 also56Run-Off Transcription and G-Less Cassette Transcription If want to assess:Transcription accuracyHow much of this accurate transcription Simpler method is run-off transcriptionCan be used after the physiological start site is found by S1 mapping or primer extensionUseful to see effects of promoter mutation on accuracy and efficiency of transcription57Run-Off 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 gene58G-Less Cassette AssayVariation of the run-off technique, instead of cutting the gene with restriction enzyme, insert a stretch of nucleotides lacking guanines in nontemplate strand just downstream of promoterAs promoter is stronger a greater number of aborted transcripts is produced59Schematic 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 autoradiography60SummaryRun-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 gel615.5 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 degradation62Nuclear Run-On TranscriptionIsolate nuclei from cells, allow them to extend in vitro the transcripts already started in vivo in a technique called run-on transcription RNA polymerase that has already initiated transcription will “run-on” or continue to elongate same RNA chainsEffective as initiation of new RNA chains in isolated nuclei does not generally occur63Run-On AnalysisResults will show transcription rates and an idea of which genes are transcribedIdentification of labeled run-on transcripts is best done by dot blottingSpot denatured DNAs on a filterHybridize to labeled run-on RNAIdentify the RNA by DNA to which it hybridizesConditions of run-on reaction can be manipulated with effects of product can be measured64Nuclear Run-On Transcription Diagram65Reporter Gene TranscriptionPlace a surrogate reporter gene under control of a specific promoter, measure accumulation of product of this reporter geneReporter 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 chemiluminescent compound that emits light66Measuring Protein Accumulation in VivoGene activity can be monitored by measuring the accumulation of protein (the ultimate gene product)Two primary methods of measuring protein accumulationImmunoblotting / Western blottingImmunoprecipitation67ImmunoprecipitationLabel proteins by growing cells with 35S-labeled amino acidBind protein of interest to an antibodyPrecipitate the protein-antibody complex with a secondary antibody complexed to Protein A on resin beads using a low-speed centrifugeDetermine protein level with liquid scintillation counting685.6 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 protein69Filter BindingFilter binding to measure DNA-protein interaction is 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 filter70Nitrocellulose Filter-Binding AssaydsDNA is labeled and mixed with proteinPour dsDNA through a nitrocellulose filterMeasure amount of radioactivity that passed through filter and retained on filter71Gel 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 protein72FootprintingFootprinting shows where a target lies on DNA and which bases are involved in protein bindingThree methods are very popular:DNase footprintingDimethylsulfate footprintingHydroxyl radical footprinting73DNase 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 gel74DMS FootprintingDimethylsulfate (DMS) is a methylating agent which can fit into DNA nooks and cranniesStarts as DNase, then methylate with DMS at conditions for 1 methylation per DNA molecule75SummaryFootprinting 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 strands765.7 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 molecule77Functional 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 activity785.8 KnockoutsProbing 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 mice79Stage 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 with altered gene and resident, intact geneThese events are rare, many cells will need to be screened using the introduced genes80Making a Knockout Mouse: Stage 181Stage 2 of the Knockout MouseIntroduce the interrupted gene into a whole mouseInject engineered cells into a mouse blastocystEmbryo into a surrogate mother who gives birth to chimeric mouse with patchy coatTrue heterozygote results when chimera mates with a black mouse to produce brown mice, half of which will have interrupted gene82Making a Knockout Mousse: Stage 283Knockout 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 mouse84

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