Bài giảng Biology - Chapter 17: Biotechnology

BiotechnologyChapter 171DNA ManipulationThe molecular biology revolution started with the discovery of restriction endonucleases -Enzymes that cleave DNA at specific sites These enzymes are significant in two ways 1. Allow a form of physical mapping that was previously impossible 2. Allow the creation of recombinant DNA molecules (from two different sources) 2DNA ManipulationRestriction enzymes recognize DNA sequences termed restriction sitesThere are two types of restriction enzymes: -Type I = Cut near the restriction site -Rarely used in DNA manipulation -Type II = Cut at the restriction site -The sites are palindromes -Both strands have same sequence when read 5’ to 3’ 3DNA ManipulationType II enzymes produce staggered cuts that generate “sticky ends” -Overhanging complementary endsTherefore, fragments cut by the same enzyme can be paired DNA ligase can join the two fragments forming a stable DNA molecule 4CCGGTATAATATCCGGTATAATATCGAATTCGAATTCGTTAACGTTAACGAATTDNA ligasejoins the strands.DNAduplexGATTCGAATTSticky endsRestriction sitesEcoRIRecombinant DNA moleculeRestriction endonucleasecleaves the DNAEcoRIEcoRIEcoRIDNA from another source cut with thesame restriction endonuclease is added.Restriction endonucleasecleaves the DNASticky endsCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.CA5Gel ElectrophoresisA technique used to separate DNA fragments by sizeThe gel (agarose or polyacrylamide) is subjected to an electrical fieldThe DNA, which is negatively-charged, migrates towards the positive pole -The larger the DNA fragment, the slower it will move through the gel matrixDNA is visualized using fluorescent dyes6Restriction endonuclease1 cut siteRestriction endonuclease2 cut siteReaction1Reaction2Reaction3Restrictionendonuclease 3Short segmentLong segmentMedium segmentMedium segmentMixture of DNAfragments ofdifferent sizes insolution placed atthe top of “lanes” inthe gelGelLaneAnode+Cathode-PowersourceShort segmentLong segmentReaction 2Reaction 1Reaction 3ShorterfragmentsLongerfragmentsVisualizing Stained GelGel is stained with a dye to allowthe fragments to be visualized.DNA samples are cut with restriction enzymes in threedifferent reactions producing different patterns of fragmentsSamples from the restriction enzyme digests are introduced into the gel.Electric current is applied causing fragments to migrate through the gel.Restriction Enzyme DigestionGel Electrophoresisa.b.c.Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.Buffer7TransformationTransformation is the introduction of DNA from an outside source into a cell Natural transformation occurs in many species -However, not in E. coli, which is used routinely in molecular biology labs -Artificial transformation techniques have been developed to introduce foreign DNA into it8Molecular CloningA clone refers to a genetically identical copyMolecular cloning is the isolation of a specific DNA sequence (usually protein-encoding) -Sometimes called gene cloningThe most flexible and common host for cloning is E. coli Propagation of DNA in a host cell requires a vector9VectorsPlasmids are small, circular extrachromosomal DNA molecules -Used for cloning small pieces of DNA -Have three important components 1. Origin of replication 2. Selectable marker 3. Multiple cloning site (MCS)10Vectors11VectorsPhage vectors are modified bacterial viruses -Most based on phage lambda (l) of E. coli -Used to clone inserts up to 40 Kbp -Have two features not shared with plasmid vectors -They kill their host cells -They have linear genomes -Middle replaced with inserted DNA12Vectors13VectorsArtificial chromosomes -Used to clone very large DNA fragments -Bacterial artificial chromosomes (BACs) -Yeast artificial chromosomes (YACs)14DNA LibrariesA collection of DNA fragments from a specific source that has been inserted into host cellsA genomic library represents the entire genomeA cDNA library represents only the expressed part of the genome -Complementary DNA (cDNA) is synthesized from isolated mRNA using the enzyme reverse transcriptase15165´ capEukaryotic DNA templateexonsintrons12341234TranscriptionPrimary RNA transcript5´ capMature RNA transcript3´ poly-A tailDegradedmRNA3´ poly-A tailmRNA–cDNA hybridIsolation of mRNAAddition of reversetranscriptaseAddition of mRNA-degrading enzymesDNA polymeraseDouble-stranded cDNAwith no intronsReversetranscriptaseReversetranscriptaseutilizes mRNAto create cDNA.Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.Introns are cut out,and coding regions arespliced together.17DNA LibrariesMolecular hybridization is a technique used to identify specific DNAs in complex mixtures -A known single-stranded DNA or RNA is labeled -It is then used as a probe to identify its complement via specific base-pairing -Also termed annealing 18DNA LibrariesMolecular hybridization is the most common way of identifying a clone in a DNA library -This process involves three steps: 1. Plating the library 2. Replicating the library 3. Screening the library19FilmFilter paper1. Colonies of plasmid containing bacteria, each containing a single DNA from the library, are grown on agar.3. The filter is washed with a solution to break the cells open and denature the DNA, which sticks to the filter at the site of each colony. The filter is incubated with a radioactively labeled probe that can form hybrids with complementary DNA in the gene of interest.4. The only sites on the filter that will retain probe DNA will contain DNA complementary to the probe. These represent the sites of colonies containing the gene of interest.5. A comparison with the original plate identifies the colony containing the2. A replica of the plate is made by pressing a piece of filter paper against the agar and bacterial colonies. Some cells from each colony adhere to the filter.20DNA AnalysisRestriction maps -Molecular biologists need maps to analyze and compare cloned DNAs -The first maps were restriction maps -Initially, they were created by enzyme digestion & analysis of resulting patterns -Many are now generated by computer searches for cleavage sites 21DNA AnalysisSouthern blotting -A sample DNA is digested by restriction enzymes & separated by gel electrophoresis -Gel is transferred (“blotted”) onto a nitrocellulose filter -Then hybridized with a cloned, radioactively-labeled DNA probe -Complementary sequences are revealed by autoradiography223. DNA in the gel is transferred, or “blotted,” onto the nitrocellulose.4. Nitrocellulose with bound DNA is incubated with radioactively labeled nucleic acids and is then rinsed.5. Photographic film is laid over the filter and is exposed only in areas that contain radioactivity (autoradiography). Bands on the film represent DNA in the gel that is complementary to the probe sequence.Size markersHybridized nucleic acidsFilmGelBufferSpongeNitrocellulose filterGel1. Electrophoresis is performed, using radioactively labeled markers as a size guide in the first lane.Test nucleicacidsRadioactivelylabeled markerswith specific sizesElectrophoreticgelElectrophoresisDNA fragmentswithin bandsRadioactiveprobe (single-stranded DNA)2. The gel is covered with a sheet of nitrocellulose and placed in a tray of buffer on top of a sponge. Alkaline chemicals in the buffer denature the DNA into single strands. The buffer wicks its way up through the gel and nitrocellulose into a stack of paper towels placed on top of the nitrocellulose.Stack of paper towels—TTACC——AATGG—SealedcontainerNitrocellulosepaper nowcontains nucleicacid “print”Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.23DNA AnalysisNorthern blotting -mRNA is electrophoresed and then blotted onto the filterWestern blotting -Proteins are electrophoresed and then blotted onto the filter -Detection requires an antibody that can bind to one protein24DNA AnalysisRFLP analysis -Restriction fragment length polymorphisms (RFLPs) are generated by point mutations or sequence duplications -These fragments are often not identical in different individuals -Can be detected by Southern blotting25a. Three different DNA duplexesb. Cut DNAc. Gel electrophoresis of restriction fragmentsOriginal Sequenceof Restriction Sites(no mutations)Point MutationsChange the Sequence of Restriction SitesSequenceRepetitions Can Occur BetweenRestriction SitesLargerfragmentsSmallerfragmentsrestriction enzymecutting sitesSequence duplicationSingle base-pairchange+++++–––Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.26DNA AnalysisDNA fingerprinting -An identification technique used to detect differences in the DNA of individuals -Makes use of a variety of molecular procedures, including RFLP analysis -First used in a US criminal trial in 1987 -Tommie Lee Andrews was found guilty of rape27DNA Analysis28DNA AnalysisDNA sequencing -A set of nested fragments is generated -End with known base -Separated by high-resolution gel electrophoresis, resulting in a “ladder” -Sequence is read from the bottom up29DNA AnalysisDNA sequencing -The enzymatic method was developed by Frederick Sanger -Dideoxynucleotides are used as chain terminators in DNA synthesis reactions30Manual Enzymatic DNA Sequencinga.GGCTAGGCTAGGGCCTTAACTAGGCCTTAAAGGCTTAATAGGCTTAGTGTGACCCCTAGCATTGTGAGAT5´3´5´5´3´Reactionfor ddGPrimerTemplateDNA polymeraseReactionfor ddCReactionfor ddAReactionfor ddTLongersegmentsShortersegments+–GCCCAAATT5´5´5´5´5´5´5´5´5´31DNA AnalysisDNA sequencing -The enzymatic technique is powerful but is labor intensive and time-consuming -The development of automated techniques made sequencing faster and more practical -Fluorescent dyes are used instead of radioactive labels -Reaction is done in one tube -Data are assembled by a computer32G5´3´PrimerTemplateDNA polymeraseGCCCAAATTManual Enzymatic DNA SequencingAutomated Enzymatic DNA Sequencinga.b.GGCTAGGCTAGGGCCTTAACTAGGCCTTAAAGGCTTAATAGGCTTAGTGTGACCCCTAGCATTGTGAGAT5´3´5´5´3´3´3´5´5´Reactionfor ddGPrimerTemplateDNA polymeraseReactionfor ddCReactionfor ddAReactionfor ddTLongersegmentsShortersegmentsGGGCCTTTTTTAAGGGCCTTTAAGGGGGGCCTTAAGGCCCCTTAAGGCTTAAAAGGCTTAGGCTAGCTACTATAALaserPhoto detectorreads colors+–GCCCAAATT5´5´5´5´5´5´5´5´5´5´5´5´5´5´5´5´5´5´5´Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.33DNA AnalysisPolymerase chain reaction (PCR) -Developed by Kary Mullis -Allows the amplification of a small DNA fragment using primers that flank the region -Each PCR cycle involves three steps: 1. Denaturation (high temperature) 2. Annealing of primers (low temperature) 3. DNA synthesis (intermediate temperature) -Taq polymerase34DNA is denaturedinto single strands5´3´5´3´3´5´3´5´Primers anneal to DNA5´3´3´5´Taq DNA polymerase5´3´5´3´3´5´3´5´5´3´5´3´5´3´5´3´5´3´5´3´5´3´5´3´3´5´3´5´3´5´3´5´3´5´3´5´3´5´3´5´5´3´5´3´5´3´5´3´3´5´3´5´3´5´3´5´PCRmachineCycle 2:4 copiesCycle 3:8 copies1. Sample is first heated to denature DNA.2. DNA is cooled to a lower temperature to allow annealing of primers.3. DNA is heated to 72°C, the optimal temperature for Taq DNA polymerase to extend primers.DNA segment to be amplified After 20 cycles, a single fragment produces over one million (220) copies!35DNA AnalysisPolymerase chain reaction (PCR) -Has revolutionized science and medicine because it allows the investigation of minute samples of DNA -Forensics -Detection of genetic defects in embryos -Analysis of mitochondrial DNA from early human species36DNA AnalysisYeast two-hybrid system -Used to study protein-protein interactions -Gal4 is a transcriptional activator with a modular structure -The Gal4 gene is split into two vectors -Bait vector: has DNA-binding domain -Prey vector: has transcription-activating domain -Neither of these alone can activate transcription37DNA AnalysisYeast two-hybrid system -When other genes are inserted into these vectors, they produce fusion proteins -Contain part of Gal4 and the protein of interest -If the proteins being tested interact, Gal4 function will be restored -A reporter gene will be expressed -Detected by an enzyme assay38FusionproteinsBait vectorBait proteinPrey vectorPrey proteinReporter geneYeast nucleusYeast cellInserted DNAInserted DNAGal4 proteinRNA polymeraseDNADNA-bindingdomainCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.Transcription-activating domainDNA-bindingdomain39Genetic EngineeringHas generated excitement and controversyExpression vectors contain the sequences necessary to express inserted DNA in a specific cell typeTransgenic animals contain genes that have been inserted without the use of conventional breeding40Genetic EngineeringIn vitro mutagenesis -Ability to create mutations at any site in a cloned gene -Has been used to produce knockout mice, in which a known gene is inactivated -The effect of loss of this function is then assessed on the entire organism -An example of reverse genetics4142Medical ApplicationsHuman proteins -Medically important proteins can be produced in bacteria -Human insulin -Interferon -Atrial peptides -Tissue plasminogen activator -Human growth hormone43Medical Applications44Medical ApplicationsVaccines -Subunit vaccines: Genes encoding a part of the protein coat are spliced into a fragment of the vaccinia (cowpox) genome -DNA vaccines: Depend on the cellular immune response (not antibodies)45Medical ApplicationsHuman immuneresponseGene specifying herpessimplex surface proteinHarmless vaccinia(cowpox) virusHerpes simplex virus2. Herpes simplex gene is isolated.3. Vaccinia DNA is extracted and cleaved.4. Fragment containing surface gene combines with cleaved vaccinia DNA.5. Harmless engineered virus (the vaccine) with surface like herpes simplex is injected into the human body.6. Antibodies directed against herpes simplex viral coat are made.1. DNA is extracted.46Medical ApplicationsGene therapy -Adding a functional copy of a gene to correct a hereditary disorder -Severe combined immunodeficiency disease (SCID) illustrates both the potential and the problems -Successful at first, but then patients developed a rare leukemia47Agricultural ApplicationsTi (tumor-inducing) plasmid is the most used vector for plant genetic engineering -Obtained from Agrobacterium tumefaciens, which normally infects broadleaf plants -However, bacterium does not infect cereals such as corn, rice and wheat48Gene ofinterestAgrobacteriumPlasmidPlant nucleus1. Plasmid is removed and cut open with restriction endonuclease.2. A gene of interest is isolated from the DNA of another organism and inserted into the plasmid. The plasmid is put back into the Agrobacterium.3. When used to infect plant cells, Agrobacterium duplicates part of the plasmid and transfers the new gene into a chromosome of the plant cell.4. The plant cell divides, and each daughter cell receives the new gene. These cultured cells can be used to grow a new plant with the introduced gene.Agricultural Applications49Agricultural ApplicationsGene guns -Uses bombardment with tiny gold particles coated with DNA -Possible for any species -However, the copy number of inserted genes cannot be controlled50Agricultural ApplicationsHerbicide resistance -Broadleaf plants have been engineered to be resistant to the herbicide glyphosate -This allows for no-till planting51Agricultural ApplicationsPest resistance -Insecticidal proteins have been transferred into crop plants to make them pest-resistant -Bt toxin from Bacillus thuringiensis Golden rice -Rice that has been genetically modified to produce b-carotene (provitamin A) -Converted in the body to vitamin A52Agricultural ApplicationsDaffodilphytoenesynthasegene (psy)psycrtIlcyPhytoenesynthaseCarotenedesaturase-CyclaseGenes introducedinto rice genomeExpressionin endospermGGPPPhytoeneLycopene-Carotene(Provitamin A)Bacterialcarotenedesaturasegene (crtI) Daffodillycopene-cyclasegene (lcy)Ricechromosome53Agricultural ApplicationsAdoption of genetically modified (GM) crops has been resisted in some areas because of questions about: -Crop safety for human consumption -Movement of genes into wild relatives -Loss of biodiversity54Agricultural ApplicationsBiopharming -Transgenic plants are used to produce pharmaceuticals -Human serum albumin -Recombinant subunit vaccines -Against Norwalk and rabies viruses -Recombinant monoclonal antibodies -Against tooth decay-causing bacteria55Agricultural ApplicationsTransgenic animal technology has not been as successful as that in plants -One interesting example is the EnviroPig -Engineered to carry the gene for the enzyme phytase -Breaks down phosphorus in feed -Reduces excretion of harmful phosphates in the environment56Agricultural Applications57