Molecular biology fourth edition - Chapter 4: Molecular cloning methods

Molecular BiologyFourth 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 links eukaryotic genes to small bacterial or phage DNAs and inserting these recombinant molecules into bacterial hostsOne can then produce large quantities of these genes in pure form2The Role of Restriction EndonucleasesRestriction endonucleases, first discovered in the late 1960s, 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 sequenceThese enzymes can recognize 4-bp, 6-bp, 8-bp sequencesThe frequency of cuts lessens when the recognition sequence is longer5Restriction Enzyme TerminologyA 6-bp cutter will yield DNA fragments averaging 4000-bp or 4 kilobases (4kb) in lengthHeteroschizomers recognize the same DNA sequence but use a different cutting site – they are also called isochizomersThese enzymes cut DNA strands reproducibly in the same place, which is extremely useful in gene analysis6Use of Restriction EndonucleasesMany restriction endonucleases make staggered cuts in the 2 DNA strandsThis leaves single-stranded overhangs, called sticky ends that can base-pair together brieflyThis makes joining 2 different DNA molecules together much easierStaggered cuts occur when the recognition sequence usually displays twofold symmetry, palindromes7Restriction-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 methylated8An Experiment Using Restriction EndonucleaseAn early experiment used EcoRI to cut 2 plasmids, small circular pieces of DNA independent of the host chromosomeEach plasmid had 1 site for EcoRICutting 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 bonds9SummaryRestriction 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 vitro10VectorsVectors function as DNA carriers to allow replication of recombinant DNAsTypical experiment uses 1 vector plus a piece of foreign DNA Depends on the vector for its replicationForeign DNA has no origin of replication, the site where DNA replication beginsThere are 2 major classes of vectors:Plasmids Phages 11Plasmids As VectorspBR plasmids were developed early but are rarely used todaypUC series is similar to pBR40% of the DNA, including tetracycline resistance has been deletedCloning sites are clustered together into one area called the multiple cloning site (MCS)12pBR322 PlasmidpBR322 illustrates cloning methods simplyResistance for 2 antibioticsTetracyclineAmpicillin Origin of replication between the 2 resistance genesOnly 1 site for several restriction enzymes13pBR322 CloningClone a foreign DNA into the PstI site of pBR322 Cut the vector to generate the sticky ends Cut foreign DNA with PstI also – compatible ends Combine vector and foreign DNA with DNA ligase to seal sticky ends Now transform the plasmid into E. coli 14Bacterial TransformationTraditional method involves incubating bacterial cells in concentrated calcium salt solutionThe solution makes the cell membrane leaky, permeable to the plasmid DNANewer method uses high voltage to drive the DNA into the cells in process called electroporation15Screening TransformantsTransformation produces bacteria with:Religated plasmidReligated insertRecombinantsIdentify the recombinants using the antibiotic resistanceGrow cells with tetracycline so only cells with plasmid grow, not foreign DNA onlyNext, grow copies of the original colonies with ampicillin which kills cells with plasmid including foreign DNA16Screening With Replica PlatingReplica plating transfers clone copies from original tetracycline plate to a plate containing ampicillinA sterile velvet transfer tool can be used to transfer copies of the original coloniesDesired colonies are those that do NOT grow on the new ampicillin plate17pUC and b-galactosidaseNewer pUC plasmids have:Ampicillin resistance geneMultiple 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 DNA18Directional CloningCut a plasmid with 2 restriction enzymes from the MCSClone in a piece of foreign DNA with 1 sticky end recognizing each enzymeThe insert DNA is placed into the vector in only 1 orientationVector religation is also prevented as the two restriction sites are incompatible19SummaryFirst generation plasmid cloning vectors include pBR322 and the pUC plasmidspBR322 has 2 antibiotic resistance genes Variety of unique restriction sites for inserting foreign DNAMost of these sites interrupt antibiotic resistance, making screening straightforwardpUC hasAmpicillin resistance geneMCS that interrupts a b-galactosidase geneMCS facilitates directional cloning into 2 different restriction sites20Phages 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 area21l Phage VectorsFirst phage vectors were constructed by Fred Blattner and colleaguesRemoved middle regionRetained genes needed for phage replicationCould replace removed phage genes with foreign DNAOriginally named Charon phageMore general term, replacement vectors22Phage Vector AdvantagesPhage vectors can receive larger amounts of foreign DNACharon 4 can accept up to 20kb of DNATraditional plasmid vectors take much lessPhage vectors require a minimum size foreign DNA piece (12 kb) inserted to package into a phage particle23Cloning Using a Phage Vector24Genomic 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 interest25Plaque HybridizationSearching a genomic library requires probe showing which clone contains desired gene Ideal probe – labeled nucleic acid with sequence matching the gene of interest26CosmidsCosmids are designed for cloning large DNA fragmentsBehave as plasmid and phageContain 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)So little phage DNA can’t replicate, but they are infectious carrying recombinant DNA into bacterial cells27M13 Phage VectorsLong, thin, filamentous phage M13Contains: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 form28M13 Cloning to Recover Single-stranded DNA ProductAfter infecting E. coli cells, single-stranded phage DNA is converted to double-stranded replicative formUse 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 29PhagemidsPhagemids are also vectorsLike cosmids have aspects of both phages and plasmidsHas a MCS inserted into lacZ’ gene to screen blue staining / 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 MCS30SummaryTwo kinds of phage are popular cloning vectorsl phage Has nonessential genes removed making room for insertsCosmids 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 libraries31Eukaryotic 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 DNA32Identifying 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 33Polynucleotide ProbesLooking for a gene you want, might use homologous gene from another organismIf already clonedHope enough sequence similarity to permit hybridizationNeed to lower stringency of hybridization conditions to tolerate some mismatches34Control of Hybridization StringencyFactors that promote separation of two strands in a DNA double helix:High temperatureHigh organic solvent concentrationLow salt concentrationAdjust conditions until only perfectly matched DNA strands form a duplex = high stringencyLowering these conditions lowers stringency until DNA strands with a few mismatches can hybridize35Protein-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 acids36SummarySpecific clones can be identified using polynucleotide probes binding to the gene itselfKnowing the amino acid sequence of the a gene product permits design of a set of oligonucleotides that encode part of the amino acid sequenceCan be a very quick and accurate means of identifying a particular clone37cDNA CloningcDNA is the abbreviation for complementary DNA or copy DNAA 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 clones38Making a cDNA Library39Reverse Transcriptase PrimerCentral to successful cloning is the synthesis of cDNA from an mRNA template using reverse transcriptase (RT), 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 primer40Ribonuclease HRT with oligo(dT) primer has made a single-stranded DNA from mRNANeed to start to remove the mRNAPartially 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 translation41Nick TranslationThe nick translation process simultaneously:Removes DNA ahead of a nickSynthesizes DNA behind nickNet result moves or translates the nick in the 5’ to 3’ directionEnzyme often used is E. coli DNA polymerase IHas a 5’ to 3’ exonuclease activity Allows enzyme to degrade DNA ahead of the nick42Trailing Terminal TransferaseDon’t have the sticky ends of genomic DNA cleaved with restriction enzymesBlunt ends will ligate, but inefficientGenerate sticky ends using 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 transformation43Vector ChoiceChoice based on method used to detect positive clonesPlasmid or phagemid like pUC or pBS will be used with colony hybridization and a labeled DNA probeIf l phage like lgt11, cloned cDNA under control of lac promoter for transcription and translation of the cloned gene and antibody screening44Rapid 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’-end455’-RACEUse 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)46SummaryMake cDNA library with synthesis of cDNAs one strand at a timeUse mRNAs from a cell as templates for 1st strands, then 1st strand as template for 2ndReverse 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’-RACE474.2 The Polymerase Chain ReactionPolymerase chain reaction (PCR) can yield a DNA fragment for cloningPCR is:More recently developedVery useful for cloning cDNAs48Standard PCRInvented by Kary Mullis and colleagues in 1980sUse 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 next round of DNA synthesisSelected region DNA now doubles in amount with each synthesis cycleSpecial heat-stable polymerases able to work after high temperatures needed to separate strands make process “set and forget” for many cycles49Amplifying DNA by PCR50Using Reverse Transcriptase (RT-PCR) in cDNA CloningTo clone a cDNA from just one mRNA whose sequence is known, use type of PCR called reverse transcriptase PCR (RT-PCR)Difference between PCR and RT-PCRStart with an mRNA not double-stranded DNABegin by converting mRNA to DNANext use forward primer to convert ssDNA to dsDNANow standard PCR continues51RT-PCR Can Generate Sticky EndsWith care, restriction enzyme sites can even be added to the cDNA of interestAble to generate sticky ends for ligation into vector of choice2 sticky ends permits directional cloning52Real-Time PCRReal-time PCR quantifies the amplification of the DNA as it occursAs DNA strands separate, anneal to forward and reverse primers, and to fluorescent-tagged oligonucleotide complementary to part of one DNA strand53Fluorescent Tags in Real-Time PCRThis fluorescent-tagged oligonucleotide serves as a reporter probeFluorescent tag at 5’-endFluorescence quenching tag at 3’-endWith PCR rounds the 5’ tag is separated from the 3’ tagFluorescence increases with incorporation into DNA product544.3 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 55Expression 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 genesFor high level expression of a cloned gene best results often with specialized expression vectorsBacterial vectors have a strong promoter and a ribosome binding site near ATG codon56Fusion 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 end57Inducible 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 promotersPrefer 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, inclusion bodies58Controlling the lac Promoterlac promoter is somewhat inducibleStays off until stimulatedActually repression is incomplete or leakySome expression will still occurTo avoid this problem, express using a plasmid or phagemid carrying its own lacI repressor gene, such as pBS59Arabinose PromoterThe hybrid trc promoter combines strength of the trp (tryptophan operon) promoter with inducibility of lac promoterPromoter from ara operon, PBAD, allow fine control of transcriptionInducible by arabinose, a sugarTranscription rate varies with arabinose concentration60Tightly Controlled PromoterLambda (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 are low temperaturesRaise temperature to nonpermissive temperature, the repressor doesn’t function and cloned gene is expressed61SummaryExpression vectors are designed to yield the protein product of a cloned geneWhen a lac inducer is added, cell begins to make T7 polymerase which transcribes the gene of interestMany molecules of T7 polymerase are made, so gene is turned on to a very high level with abundant amount of protein product made62Expression 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 Ni2+ Permits purification by nickel affinity chromatographyHis tag can be removed using enzyme enterokinase without damage to the protein product63Oligohistidine Expression Vector64Fusion Proteins in lgt11This phage contains lac control region and lacZ geneProducts of gene correctly inserted will be fusion proteins with a b-galactosidase leader65Antibody Screening With lgt11Lambda phages with cDNA inserts are platedProtein released are blotted onto a supportProbe with antibody to proteinAntibody bound to protein from plaque is detected with labeled protein APartial cDNAs can be completed with RACE66SummaryExpression vectors frequently produce fusion proteinsOne part of the protein comes from coding sequences in the vectorOther part from sequences in the cloned geneMany fusion proteins have advantage of being simple to isolate by affinity chromatographyVector lgt11 produces fusion proteins that can be detected in plaques with a specific antiserum67Bacterial Expression System ShortcomingsThere are problems with expression of eukaryotic proteins in a bacterial systemBacteria may recognize the proteins as foreign and destroy themPosttranslational modifications are different in bacteriaBacterial environment may not permit correct protein foldingVery high levels of cloned eukaryotic proteins can be expressed in useless, insoluble form68Eukaryotic Expression SystemsAvoid bacterial expression problems by expressing the protein in 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 cultureStill a eukaryote with more appropriate posttranslational modificationSecretes protein in growth medium so easy purification69Use 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 cellsPromoter 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 cannot result in infectious virus as it lacks an essential gene supplied by the vector70Baculovirus Expression71Animal Cell TransfectionCalcium phosphateMix cells with DNA in a phosphate bufferThen solution of calcium salt added to form a precipitateCells take up the calcium phosphate crystals which include some DNALiposomesDNA mixed with lipid to form liposomes, small vesicles with some of the DNA insideDNA-bearing liposomes fuse with cell membrane carrying DNA inside the cell72SummaryForeign genes can be expressed in eukaryotic cellsThese eukaryotic systems have advantages over prokaryotic onesMade in eukaryotic cells tend to fold properly and are then soluble rather than aggregated into insoluble inclusion bodiesPosttranslational modifications are made in a eukaryotic manner73Using 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 galls74Ti 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 plant75Ti Plasmid Transfers Crown Gall76Use of the T-DNA Plasmid77