Bài giảng Molecular Biology - Chapter 4 Molecular Cloning Methods

Molecular BiologyFifth EditionChapter 4Molecular Cloning MethodsLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.14.1 Gene CloningGene cloning is an indispensable molecular biology technique that allows scientists to produce large quantities of their gene of interestGene cloning links eukaryotic genes to small bacterial or phage DNAs and inserting these recombinant molecules into bacterial hostsGene cloning can produce large quantities of these genes in pure form2The Role of Restriction EndonucleasesRestriction endonucleases, first discovered in the late 1960s in E. coli, are named for preventing invasion by foreign DNA by cutting it into piecesThese enzymes cut at sites within the foreign DNA instead of chewing from the endsBy cutting DNA at specific sites they function as finely honed molecular knives3Naming Restriction EndonucleasesRestriction endonucleases are named using the 1st three letters of their name from the Latin name of their source microorganism Hind IIIFirst letter is from the genus H from HaemophilusNext two letters are the 1st two letters of the species name in from influenzaeSometimes the strain designation is included “d” from strain RdIf microorganism produces only 1 restriction enzyme, end the name with Roman numeral I Hind IIf more than one restriction enzyme is produced, the others are numbered sequentially II, III, IV, etc.4Restriction Endonuclease SpecificityRestriction endonucleases recognize a specific DNA sequence, cutting ONLY at that sequenceThey recognize 4-bp, 6-bp, 8-bp palindromic sequencesThe frequency of cuts lessens as the recognition sequence is longerThey cut DNA reproducibly in the same place5Restriction-Modification SystemWhat prevents these enzymes from cutting up the host DNA?They are paired with methylasesTheses enzymes recognize, methylate the same siteTogether they are called a restriction-modification system, R-M systemMethylation protects DNA, after replication the parental strand is already methylated6An Experiment Using Restriction Endonuclease: Boyer and Cohen An early experiment used EcoRI to cut 2 plasmids, small circular pieces of DNA independent of the host chromosomeEach plasmid had 1 EcoRI siteCutting converted circular plasmids into linear DNA with the same sticky endsThe ends base pairSome ends re-closeOthers join the 2 piecesDNA ligase joins 2 pieces with covalent bonds7SummaryRestriction endonucleases recognize specific sequences in DNA molecules and make cuts in both strandsThis allows very specific cutting of DNAsThe cuts in the two strands are frequently staggered, so restriction enzymes can create sticky ends that help to link together 2 DNAs to form a recombinant DNA in vitro8VectorsVectors function as DNA carriers to allow replication of recombinant DNAsTypical experiment uses 1 vector plus a piece of foreign DNA The inserted and foreign DNA depends on the vector for its replication as it does not have an origin of replication, the site where DNA replication beginsThere are 2 major classes of vectors:Plasmids Phages 9Plasmids As VectorspBR plasmids were developed early but are rarely used todaypUC series is similar to pBR40% of the DNA has been deletedCloning sites are clustered together into one area called the multiple cloning site (MCS)MCS allows one to cut the vector and foreign gene with two different restriction enzymes and use a directional cloning technique to know the orientation of the insert10Screening: antibiotics and b-galactosidaseScreening capabilities within plasmids:Antibiotic resistance genes (i.e., ampicillin resistance gene) allow for the selection of bacteria that have received a copy of the vectorMultiple cloning site inserted into the gene lacZ’ coding for the enzyme b-galactosidaseClones with foreign DNA in the MCS disrupt the ability of the cells to make b-galactosidasePlate on media with a b-galactosidase indicator (X-gal) and clones with intact b-galactosidase enzyme will produce blue coloniesColorless colonies should contain the plasmid with foreign DNA compared to blue colonies that do not contain the plasmid with DNA11SummaryFirst generation plasmid cloning vectors include pBR322 and the pUC plasmidsScreening capabilities:Ampicillin resistance geneMCS that interrupts a b-galactosidase geneMCS facilitates directional cloning into 2 different restriction sites for orientation of inserted gene12Phages As VectorsBacteriophages are natural vectors that transduce bacterial DNA from one cell to anotherPhage vectors infect cells much more efficiently than plasmids transform cellsClones are not colonies of cells using phage vectors, but rather plaques, a clearing of the bacterial lawn due to phage killing the bacteria in that area13l Phage VectorsFirst phage vectors were constructed by Fred Blattner and colleaguesModifications included removal of the middle region and retention of the genes needed for phage replicationCould replace removed phage genes with foreign DNAAdvantage: Phage vectors can receive larger amounts of foreign DNA (up to 20kb of DNA)Traditional plasmid vectors take much lessPhage vectors require a minimum size foreign DNA piece (12 kb) inserted to package into a phage particle14Cloning Using a Phage Vector15Genomic LibrariesA genomic library contains clones of all the genes from a species genomeRestriction fragments of a genome can be packaged into phage using about 16 – 20 kb per fragmentThis fragment size will include the entirety of most eukaryotic genesOnce a library is established, it can be used to search for any gene of interest16Selection via Plaque HybridizationSearching a genomic library requires a probe to determine which clone contains the desired gene Ideal probe – labeled nucleic acid with sequence matching the gene of interest17CosmidsCosmids are designed for cloning large DNA fragmentsBehave both as plasmid and phage and contain cos sites, cohesive ends of phage DNA that allow the DNA to be packaged into a l phage headPlasmid origin of replication permitting replication as plasmid in bacteriaNearly all l genome removed so there is room for large inserts (40-50 kb)Very little phage DNA yields them unable to replicate, but they are infectious and carry their recombinant DNA into bacterial cells18M13 Phage VectorsLong, thin, filamentous phageContains:Gene fragment with b-galactosidaseMultiple cloning site like the pUC familyAdvantageThis phage’s genome is single-stranded DNAFragments cloned into it will be recovered in single-stranded form19M13 Cloning to Recover Single-stranded DNA ProductAfter infecting E. coli cells, single-stranded phage DNA is converted to double-stranded replicative form (RF)Use the replicative form for cloning foreign DNA into MCSRecombinant DNA infects host cells resulting in single-stranded recombinant DNAPhage particles, containing single-stranded phage DNA is secreted from transformed cells and can be collected from media 20PhagemidsPhagemids are also vectorsLike cosmids have aspects of both phages and plasmidsHas MCS inserted into lacZ’ gene to screen blue/ white coloniesHas origin of replication of single-stranded phage f1 to permit recovery of single-stranded recombinant DNAMCS has 2 phage RNA polymerase promoters, 1 on each side of MCS21SummaryTwo kinds of phage are popular cloning vectorsl phage Has nonessential genes removed making room for inserts up to 20kbCosmids can accept DNA up to 50 kbM13 phageHas MCSProduces single-stranded recombinant DNAPlasmids called phagemids also produce single-stranded DNA in presence of helper phageEngineered phage can accommodate inserts up to 20 kb, useful for building genomic libraries22Eukaryotic Vectors and Very High Capacity VectorsThere are vectors designed for cloning genes into eukaryotic cellsOther vectors are based on the Ti plasmid to carry genes into plant cellsYeast artificial chromosomes (YAC) and bacterial artificial chromosomes (BAC) are used for cloning huge pieces of DNA23Identifying a Specific Clone With a Specific ProbeProbes are used to identify a desired clone from among the thousands of irrelevant onesTwo types are widely usedPolynucleotides (also called oligonucleotides)Antibodies 24Polynucleotide ProbesLooking for the gene you want, you might use the homologous gene from another organismIf already cloned and there is enough sequence similarity to permit hybridizationNeed to lower stringency of hybridization conditions to tolerate some mismatchesHigh temperature, high organic solvent concentration and low salt concentration are factors that promote separation of two strands in a DNA double helix and can be adjusted as needed25Protein-based Polynucleotide ProbesNo homologous DNA from another organism?If amino acid sequence is known, deduce a set of nucleotide sequences to code for these amino acidsConstruct these nucleotide sequences chemically using the synthetic probesWhy use several?Genetic code is degenerate with most amino acids having more than 1 nucleic acid tripletMust construct several different nucleotide sequences for most amino acids26SummarySpecific clones can be identified using polynucleotide probes binding to the gene itselfKnowing the amino acid sequence of the gene product permits design of a set of oligonucleotides that encode part of the amino acid sequenceThis can be a very quick and accurate means of identifying a particular clone27cDNA CloningcDNA - complementary DNA or copy DNA that is a DNA copy of RNAA cDNA library is a set of clones representing as many as possible of the mRNAs in a given cell type at a given timeSuch a library can contain tens of thousands of different clones28Making a cDNA Library29Reverse TranscriptaseCentral to successful cloning is the synthesis of cDNA from an mRNA template using reverse transcriptase (RT), an RNA-dependent DNA polymeraseRT cannot initiate DNA synthesis without a primerUse the poly(A) tail at 3’ end of most eukaryotic mRNA so that oligo(dT) may serve as primer30Ribonuclease HRT with oligo(dT) primer has made a single-stranded DNA off of mRNANeed to remove the RNAPartially degrade the mRNA using ribonuclease H (RNase H)Enzyme degrades RNA strand of an RNA-DNA hybridRemaining RNA fragments serve as primers for “second strand” DNA using nick translation31Nick TranslationThe nick translation process simultaneously:Removes DNA ahead of a nickSynthesizes DNA behind nickNet result moves the nick in the 5’ to 3’ directionEnzyme often used is E. coli DNA polymerase IHas 5’ to 3’ exonuclease activity Allows enzyme to degrade DNA ahead of the nick32Terminal TransferasecDNAs don’t have the sticky ends of genomic DNA cleaved with restriction enzymesBlunt ends will ligate, but is inefficientGenerate sticky ends using enzyme terminal deoxynucleotidyl transferase (TdT), terminal transferase with one dNTPIf use dCTP with the enzymedCMPs are added one at a time to 3’ ends of the cDNASame technique adds oligo(dG) ends to vectorGenerate ligation product ready for transformation33Rapid Amplification of cDNA EndsIf generated cDNA is not full-length, missing pieces can be filled in using rapid amplification of cDNA ends (RACE)Technique can be used to fill in either the missing portion at the 5’-end (usual problem)Analogous technique can be used to fill in a missing 3’-end34RACE ProcedureUse RNA prep containing mRNA of interest and the partial cDNAAnneal mRNA with the incomplete cDNAReverse transcriptase will copy rest of the mRNATail the completed cDNA with terminal transferase using oligo(dC)Second strand synthesis primed with oligo(dG)35SummarycDNA library can be synthesized using mRNAs from a cell as templates for the 1st strands that is then used as a template for the 2nd strandsReverse transcriptase generates 1st strandDNA polymerase I generates the second strandsGive cDNAs oligonucleotide tails that base-pair with complementary tails on a cloning vectorUse these recombinant DNAs to transform bacteriaDetect clones with:Colony hybridization using labeled probesAntibodies if gene product translatedIncomplete cDNA can be filled in with 5’- or 3’-RACE364.2 The Polymerase Chain ReactionPolymerase chain reaction (PCR) is used to amplify DNA and can be used to yield a DNA fragment for cloningInvented by Kary Mullis and colleagues in 1980sSpecial heat-stable polymerases are now used that are able to work after high temperatures - researchers no longer need to add fresh DNA polymerase after each round of replication37Standard PCRUse enzyme DNA polymerase to copy a selected region of DNAAdd short pieces of DNA (primers) that hybridize to DNA sequences on either side of piece of interest – causes initiation of DNA synthesis through that area, XCopies of both strands of X and original DNA strands are templates for the next round of DNA synthesisThe selected region of DNA now doubles in amount with each synthesis cycle38Amplifying DNA by PCR39Using Reverse Transcriptase PCR (RT-PCR) in cDNA CloningTo clone a cDNA from just one mRNA whose sequence is known, a type of PCR called reverse transcriptase PCR (RT-PCR) can be usedDifference between PCR and RT-PCRStarts with an mRNA, not dsDNABegin by converting mRNA to DNAUse forward primers to convert ssDNA to dsDNAContinue with standard PCR40RT-PCR to clone a single cDNAWith care, restriction enzyme sites can even be added to the ends of the cDNA of interestAble to generate sticky ends for ligation into vector of choice2 sticky ends permits directional cloning41Real-Time PCRReal-time PCR quantifies the amplification of the DNA as it occursAs the DNA strands separate, they anneal to forward and reverse primers, and to a fluorescent-tagged oligonucleotide complementary to part of one DNA strand that serves as a reporter probe42Real-Time PCRA fluorescent-tagged oligonucleotide serves as a reporter probeFluorescent tag at 5’-endFluorescence quenching tag at 3’-endAs PCR progresses from the forward primer the 5’ tag is separated from the 3’ tag and allows the 5’ tag to fluoresceFluorescence increases with incorporation into DNA product and can be quantitated434.3 Methods of Expressing Cloned GenesCloning a gene permits Production of large quantities of a particular DNA sequence for detailed studyLarge quantities of the gene’s product can also be obtained for further useStudy Commerce 44Expression VectorsVectors discussed so far are used to first put a foreign DNA into a bacterium to replicate and screenExpression vectors are those that can yield protein products of the cloned genesBacterial expression vectors typically have two elements required for active gene expression; a strong promoter and a ribosome binding site near an initiating codon45Fusion ProteinsSome cloning vectors, pUC and pBS, can work as expression vectors using lac promoterIf inserted DNA is in the same reading frame as interrupted gene, a fusion protein resultsThese have a partial b-galactosidase sequence at amino endInserted cDNA protein sequence at carboxyl end46Inducible Expression VectorsMain function of expression vector is to yield the product of a gene – usually more is betterFor this reason, expression vectors have very strong promotersIt is usually advantageous to keep a cloned gene repressed until time to expressLarge quantities of eukaryotic protein in bacteria are usually toxicCan accumulate to levels that interfere with bacterial growthExpressed protein may form insoluble aggregates, called inclusion bodies47Controlling the lac Promoterlac promoter is somewhat inducibleStays off until stimulated by inducer IPTGHowever, repression is typically incomplete or leaky and some expression will still occurTo avoid this problem, use a plasmid or phagemid carrying its own lacI repressor gene to keep the cloned gene off until it is induced by IPTG48Alternatives to the lac PromoterThe hybrid trc promoter combines the strength of the trp (tryptophan operon) promoter with the inducibility of the lac promoterPromoter from ara operon, PBAD, allow fine control of transcriptionInducible by arabinose, a sugarTranscription rate varies with arabinose concentration49The lambda (l) phage promoter, PL, is tightly controlledExpression vectors with this promoter-operator system are used in host cells with temperature-sensitive l repressor geneRepressor functions at low temperaturesRaise temperature above the nonpermissive level (42’C) and the repressor doesn’t function and the cloned gene is expressedAlternatives to the lac Promoter50SummaryExpression vectors are designed to yield the protein product of a cloned geneTo optimize expression, these vectors include strong bacterial or phage promoters and bacterial ribosome binding sitesMost cloning vectors are inducible, which avoids premature overproduction of a foreign product that could poison the bacterial host cells51Expression Vectors That Produce Fusion ProteinsMost vectors express fusion proteinsThe actual natural product of the gene isn’t madeExtra amino acids help in purifying the protein productOligohistidine expression vector has a short sequence just upstream of MCS encoding 6 HisOligohistidine has a high affinity for divalent metal ions like nickel (Ni2+) Permits purification by nickel affinity chromatographyThe his tag can be removed using enzyme enterokinase without damage to the protein product52Using an Oligohistidine Expression Vector53Expression vector lgt11This phage contains the lac control region followed by the lacZ geneThe cloning sites are located within the lacZ geneProducts of gene correctly inserted will be fusion proteins with a b-galactosidase leader54Detecting positive lgt11 clones via antibody screeningLambda phages with cDNA inserts are platedProtein released are blotted onto a supportProbe with antibody specific to proteinAntibody bound to protein from plaque is detected with labeled protein A55SummaryExpression vectors frequently produce fusion proteins with one part of the protein coming from the coding sequences in the vector and the other part from sequences in the cloned geneMany fusion proteins have advantage of being simple to isolate by affinity chromatographyVector gt11 produces fusion proteins that can be detected in plaques with a specific antiserum56Bacterial Expression System ShortcomingsThere are problems with expression of eukaryotic proteins in a bacterial systemBacteria may recognize the proteins as foreign and destroy themPost-translational modifications are different in bacteriaBacterial environment may not permit correct protein foldingVery high levels of cloned eukaryotic proteins can be expressed in useless, insoluble form57Eukaryotic Expression SystemsAvoid bacterial expression problems by expressing the protein in a eukaryotic cellInitial cloning done in E. coli using a shuttle vector, able to replicate in both bacterial and eukaryotic cellsYeast is suited for this purposeRapid growth and ease of cultureA eukaryote with more appropriate post-translational modificationUse of the yeast export signal peptide secretes protein into growth medium for easy purification58Use of Baculovirus As Expression VectorViruses in this class have a large circular DNA genome, 130 kbMajor viral structural protein is made in huge amounts in infected cellsThe promoter for this protein, polyhedrin, is very activeThese vectors can produce up to 0.5 g of protein per liter of mediumNonrecombinant viral DNA entering cells does not result in infectious virus as it lacks an essential gene supplied by the vector59Expressing a Gene in a Baculovirus60Animal Cell TransfectionCarried out in two ways:Calcium phosphateMix cells with DNA in a phosphate buffer and add a solution of calcium salt to form a precipitateThe cells take up the calcium phosphate crystals, which include some DNALiposomesThe DNA is mixed with lipid to form liposomes, small vesicles with some of the DNA insideDNA-bearing liposomes fuse with the cell membrane to deliver DNA inside the cell61SummaryForeign genes can be expressed in eukaryotic cellsThese eukaryotic systems have advantages over prokaryotic systems for producing eukaryotic proteinsThe proteins tend to fold properly and are soluble, rather than aggregated into insoluble inclusion bodiesPost-translational modifications are compatible62Using the Ti Plasmid to Transfer Genes to PlantsGenes can be introduced into plants with vectors that can replicate in plant cellsCommon bacterial vector promoters and replication origins are not recognized by plant cellsPlasmids are used containing T-DNAT-DNA is derived from a plasmid known as tumor-inducing (Ti)Ti plasmid comes from bacteria that cause plant tumors called crown galls63Ti Plasmid InfectionBacterium infects plant, transfers Ti plasmid to host cellsT-DNA integrates into the plant DNA causing abnormal proliferation of plant cellsT-DNA genes direct the synthesis of unusual organic acids, opines which can serve as an energy source to the infecting bacteria but are useless to the plant64The Ti Plasmid Transfers Crown Gall65Use of the T-DNA Plasmid66SummaryMolecular biologists can transfer cloned genes to plants, creating transgenic organisms with altered characteristics, using a plant vector such as the Ti plasmid67