An Abandoned Presentation for Sure Shot Cloning Atul Kakrana

ASeminar Discussion onPCR andCloningofInsertDidanybodysaid CLONING?

By, Atul Kakrana [email protected]/24/2010 AnabandonedpresentationforSURESHOT CLONING AtulKakrana

Prologue A1971paperintheJournalofMolecularBiologybyKleppeandNobellaureateH.GobindKhoranafirst describedamethodusinganenzymaticassaytoreplicateashortDNAtemplatewithprimersinvitro ThisearlymanifestationofthebasicPCRprincipledidnotreceivemuchattention,andtheinventionofthe polymerasechainreactionin1983isgenerallycreditedtoKaryMullis. MullisreceivedtheNobelprizeinchemistry (1993)forhisdevelopmentofthe PolymeraseChain Reaction (PCR)

PCRBASEDCLONING CloningPCRproductsintoplasmidvectorsisacommondownstreamapplicationofPCR. CommonlyusedstrategyforPCRcloningistoaddrestrictionenzymerecognitionsitestotheendsofPCR primers.ThePCRproductisthendigestedandclonedintothedesiredvector.

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AnabandonedpresentationforSURESHOT CLONING AtulKakrana

PCR Nostrum for Science and Research Thepolymerasechainreaction(PCR)isatechniquetoamplifyasingleorfewcopiesofapieceofDNAacross severalordersofmagnitude,generatingthousandstomillionsofcopiesofaparticularDNAsequence. Themethodreliesonthermalcycling,consistingofcyclesofrepeatedheatingandcoolingofthereactionforDNA meltingandenzymaticreplicationoftheDNA.

TheProgram: InitialHeating AsteprequiredonlyforheatactivationincaseofHotstartPCR.9498C/19min TheCycle: Denaturation :ItcausesmeltingoftheDNAtemplatebydisruptingthehydrogenbondsbetween complementarybases,yieldingsinglestrandsofDNA.9496 C/2030Sec Annealing: ItallowsannealingoftheprimerstothesinglestrandedDNAtemplate.5565 C/2040Sec Polymerization:InthissteptheDNApolymerasesynthesizesanewDNAstrandcomplementarytotheDNA templatestrandbyaddingdNTPs thatarecomplementarytothetemplatein5'to3'direction.The temperaturedependsuponthepolymeraseusedi.e simpleTaq polymerasehasoptimumactivityat72 C. FinalPolymerization:ThissinglestepisoccasionallyperformedafterthelastPCRcycletoensurethatany remainingsinglestrandedDNAisfullyextended.7074 C/515min.

Hold:Thisistheoptionalmost important stepofthewholereaction(Givesyoufreedomtosleepoveryour PCR).4-8 C

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Polymerase Chain Reaction

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Optimizing PCR Annealing Temperature : Run a Gradient PCR with increments of 2 C from 55 -65 C. If product composition is known then annealing temperature can also be calculated with relation TaOpt = 0.3TmPrimer + 0.7TmProduct - 14.9 (W.Rychlik et al., 1990). DNA Polymerase : The lack in 3' to 5' proofreading of the Taq polymerase enzyme results in a high error rate (mutations per nucleotide per cycle) of approximately 1 in 9,000 bases, which affects the fidelity of the PCR. It is recommended to use High Fidelity polymerase for cloning and sequencing needs. Magnesium Concentration : Magnesium is required as a cofactor for DNA polymerase. Inadequate thawing of MgCl2 may result in the formation of concentration gradients within the magnesium chloride solution and contribute to failed experiments. Low Mg conc. will lead to non specific products whereas high mg concentration will inhibit DNA polymerase activity. Optimal conc. Is 1.83.6mM. Deoxynucleotides : Excessive amounts of dNTPs can increase the error rate of DNA polymerase and even inhibit the reaction. An imbalance in the proportion of the four dNTPs can result in misincorporation into the newly formed DNA strand and contribute to a decrease in the fidelity of DNA polymerase

Contamination : To avoid contamination during setting up of PCR, filter tips and hand gloves should be used. Laminar hood can also be used to set up a PCR. Negative Control is recommended to check contamination during handling. Control reaction is set up in the same way as the experimental PCRs, but without template DNA added, and is performed alongside the experimental PCRs.

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Troubleshooting PCRPROBLEM NoAmplicon CAUSES Incorrectannealingtemperature Primerdimers LowYield Annealingtemperaturenotoptimal Insufficientcycles Extensiontimetooshort Longdenaturation step,inactivating theenzyme NonSpecific Amplification MultipleProducts Primingstartingduringsetup SOLUTIONS Run a temperature gradient in 2C increments Increase temperature and/or decrease MgCl2. Check self complementarity of primers. Run a temperature gradient in 2C increments Increase amount of cycles For long products (>2kb), extension time (in mins) should be approximately equal to the number of kb in the amplicon. Use 2 minute denaturation time for polymerases which do not require a hotstart. Set up reaction on ice or use a hotstart Taq polymerase

Annealingtemperaturenotoptimal Primersnotspecific Annealingtimetoolong

Run a temperature gradient in 2C increments Blast primers to check specificity. Redesign primers Decrease time of annealing step

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CONTD. PROBLEMNonSpecificAmplification SmearedProduct

CAUSESPrimingstartingduringsetup Annealingtemperaturenotoptimal Templatedegraded

SOLUTIONSSetupreactiononiceoruseahotstartTaq polymerase Runatemperaturegradientin2Cincrements MinimizefreezethawingofDNA.Runtemplate onagarose geltocheckintegrity. Forlongproducts(>2kb),extensiontime(in mins)shouldbeapproximatelyequaltothe numberofkbintheamplicon. dNTPs areverysusceptibletofreezethawing. Replacewithafreshaliquot Checkanynewcomponentsthathavebeen added(eg.newbatchofprimers)

Extensiontimetooshort

ReactionNotReproducibleor ReactionStoppedWorking

dNTPs degraded Changeincomponent

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Be specific! The conventional Taq DNA polymerase is active at room temperature and to a lesser degree, even on ice When all the reaction components are put together, nonspecific primer annealing can occur due to these low temperatures Nonspecifically annealed primer can then be extended by the Taq DNA polymerase, generating nonspecific products and lowering product yields BESPECIFICWITHYOURPRODUCTS USEHOTSTART HotstartPCR techniqueutilizesspecializedDNApolymerasesthatgetsactivatedonlyathigh temperature AnantibodyorinhibitorisboundtoDNApolymerasethatdenaturesanddissociatesathightemperature, restoringDNApolymeraseactivity. Thisinhibitsanynonspecificextensionofannealedprimersatroomtemperature NEEDULITIMATESPECIFICITYANDYIELD TRY HOTSTART+TOUCHDOWNPCR Touchdown PCR circumvents spurious priming by starting with highest possible annealing temperature (Just below Tm) and lowering it with subsequent set of cycles. Highly specific binding takes place initially at high temperature and exponential nature of PCR increases the copy of specific sequences thereby outcompeting the non specific sequences to which primers may bind at following lower temperatures.AnabandonedpresentationforSURESHOT CLONING AtulKakrana

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Primer DesigningThingstoknowbeforeprimerdesigning PrimerLength:1820bpoflengthislongenoughforadequatespecificity.Longerprimerswillhavehigh TmandwillgivedifficultiesduringPCRoptimization. PrimerMeltingtemperature: GenerallyTmshouldbebelow65Ctoavoidsecondaryannealing.Primers withmeltingtemperatureintherangeof5862Cproducegoodresults. GCContent: TheGCcontent(thenumberofG'sandC'sintheprimerasapercentageofthetotalbases) ofprimershouldbe4060%. GCClamp:ThepresenceofGorCbaseswithinthelastfivebasesfromthe3'endofprimers(GCclamp) helpspromotespecificbindingatthe3'endduetothestrongerbondingofGandCbases. SecondaryStructures:1. 2. 3. SelfDimers:Aprimerselfdimerisformedbyintermolecularinteractionsbetweenthetwo(samesense)primers, wheretheprimerishomologoustoitself.ToleratedwithinGof56kcal/mol. CrossDimer:Primercrossdimersareformedbyintermolecularinteractionbetweensenseandantisenseprimers, wheretheyarehomologous.ToleratedwithinGof56kcal/mol. Hairpins:Itisformedbyintramolecularinteractionwithintheprimerandshouldbeavoided.ToleratedwithinGof 23kcal/mol.

RepeatsandRuns: Primerswithlongrunsofasinglebaseshouldgenerallybeavoidedastheycanmis prime,ex ACGTAAAAAAT.Runsof4bparetolerable.Similarly,repeatisadinucleotideoccurringmany timesconsecutivelyandshouldbeavoided.Repeatsof4dinucleotidesareacceptable CrossHomology:Blasttheprimerswiththedatabaseoforganismofinteresttocheckthespecificityof primers. AnabandonedpresentationforSURESHOT SCREENCASTFORPRIMERDESIGNINGFOLLOWSNEXT 5/24/2010 CLONING AtulKakrana

Primer 3 : Design Primers (Screencast)

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Inclusion of restriction sites in primers for directional cloningRESTRICTIONSITEANALYSISOFINSERTANDVECTOR Which sites are available in vector of interests? Check Vector map Which sites are already present in fragment of interest? Use freely available software's such as BioEdit or Emboss suite to find all restriction sites present in the target sequence. JUMP TO NEXT SLIDE TO CHECK Avoid sites that are common in both vector and insert and choose among other sites with these criteria's in mind:1. 2. 3. 4. 5. Enzymes for selected sites should have a compatible buffer for efficient double digestion to expedite cloning Enzymes should have heat inactivation option to avoid gel elution before ligation Restriction sites should be spaced apart (at least 10bp), this improves the efficiency of double digestion Both the selected sites should produce sticky ends for directional cloning, one blunt and one sticky will also lead to directional cloning Spacer nucleotides have to be added at 5 end just after the restriction sites. Every restriction site demands different number of spacer nucleotides. Minimum number of base pairs required for efficient restriction can be checked on http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_olignucleotides.asp

Add selected sites one in each at 5 end of forward and reverse primers. Restriction sites should be included in such a way that orientation of insert is in conjugation with vector. An Example of primer sets with restriction sites: Spacer + Res. Site + Primer ForwardACACGGATCCGTATTGAAGAACGTTTGCGACTG|58.7C|BamHI ReverseTAATGTCGACCCGGCGGAAGGGAATGAAG|58.7C|SalI

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Analyze your sequence for restriction sites (Screencast)

Analyzevectorforcommonsites

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Restriction of Vector and InsertTipsfordoubledigestion Keepglycerolconcentrationlessthen5%inareaction,toavoidthisrestrictionenzymeconc.Shouldnot bemorethen1/10ofreactionvolume. DNApreparationsmayhaveimpuritieswhichcaninhibitrestrictionenzymedigestionactivity.SomeDNA isolationkitsevenusehighEDTAbufferstoelutetheDNA.PurifyyourDNAeitherbygelelutionorPCR purificationkit. Setupacontroldigestwhenusingrestrictionenzymes.UseDNAwhichgeneratesknownfragmentation patterns.

DigestionofVectorandInsertwithselected(two)restrictionsites Digest both vector and insert with same restriction sites to favor directional cloning. Heat inactivation (raising the temperature to 65 or 80C for 20 minutes) is the simplest method of stopping a reaction. Heat inactivation does not work for all restriction enzymes. Phenol/chloroform extraction, gel elution or commercial kits can be used to purify the insert.AnabandonedpresentationforSURESHOT CLONING AtulKakrana

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Troubleshooting your digestion reactionPROBLEMIncompleteorNo digestion

CAUSEEnzymeisinactive Reactionconditionsarenot optimal DNAconcentrationisnot optimal Recognitionsitemaybetoo closetotheendoftheDNA fragment

SOLUTIONTestenzymeoncontrolDNAwithknownmultiple sites Followrecommendationsfordoubledigestion,or tryasequentialdigest Recommended conc.Is 1gofDNAina50l reaction. ExcessDNAmayresultinincompletecleavage Asageneralrule,add6basespairsoneitherside oftherecognitionsite PrepareanewDNAsample

UnexpectedCleavage pattern

DNAsampleiscontaminated

Additionalrecognitionsitesare ConfirmDNAsequence presentinDNA StarActivity Choosecorrectbuffer/Reduce theincubationtime

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Finally! Eligible for ligationThe mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3' hydroxyl ends of one nucleotide with the 5' phosphate end of another. which can be catalyzed by two different ligases: E. coli DNA ligase and bacteriophage T4 DNA ligase. The latter is the preferred enzyme because it can also join bluntended DNA fragments. Tipsforefficientligation Before ligation, completely inactivate restriction enzyme by heat inactivation, spin column or Phenol/EtOH purification Keep total DNA concentration between 110 g/ml i.e 10 100ng/10l reaction mix Insert: Vector molar ratios between 2:1 and 6:1 are optimal for single insertions Quantify your restricted vector and insert before ligation step to save hard labor. If you are unsure of your DNA concentration, perform multiple ligations with varying ratios Ligase buffer contains ATP, which is unstable and degraded by multiple freeze/thaw cycles. Make 1020ul aliquots from the original Do not heat inactivate ligase if there is PEG in the reaction buffer because transformation will be inhibited. Few Commercially available kits contains PEG5/24/2010

Anexampleofligation(stickyends)


A yummy ligation recipeIngredients :Quantifiedvectorandinsert(restricted),T4Ligase ,BufferandNucleasefreewater Select/Chooseyourmolarratio,generallyVector:Insertratioof1:3ispreferred Usethisformulatocalculateamountofvectorandinsertrequiredforselectedmolarratio: Insertmass(ng)=6X[insertlength(bp)/vectorlength(bp)]XVectormass(ng) Alternativelyonlineligationcalculatorscanalsobeusedtocalculatevectorandinsertamount: http://www.promega.com/biomath/calc06.htm (Promega) http://www.fermentas.com/reviewer/app?page=Calculator&service=external&sp=Sligations (Fermentas) http://www.insilico.uniduesseldorf.de/Lig_Input.html (HeinrichHeineUniversitt)

Setupyourligationmixforatotalvolumeof510l.Thisvolumeisenoughforsuccessfultransformation andsavesyouringredientstoo.

Incubation: Incubateovernightattemperaturerecommendedbytheenzymemanufacturer.Moreunitsof ligase canbeaddedtoshortenthetimeofincubation.Increaseinligationtemperaturealsoshortensthe timeofincubationrequired.

SCREENCASTFORLIGATIONCALCULATIONFOLLOWS

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Calculate your ligation mix (Screencast)

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Troubleshooting : LigationPROBLEM NoLigationat all CAUSE Insert/vectoraredegradedpriorto or duringligation Faultyrestriction Componentofligationismissingor not working OverdigestionofVectorandinsert(Star activity) Hiddenrestrictionsite Theligase wasinactive Decreasedligation efficiency Insufficient DNAeithervectororinsert highsaltorEDTAinthereaction SOLUTION CheckDNAonGel Transform cutanduncutvectorand lookforcolonies Alwaysputpositivecontrol Useofexcessofrestrictionenzymeand prolongedincubationshouldbe avoided Analyze targetsequenceforall restrictionsites TestonlambdaHindIII orother convenientsubstrate Increaseinsert/vectorratio CleanuptheDNA

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TransformationTransformation is the genetic alteration of a cell resulting from the uptake, genomic incorporation, and expression of environmental genetic material (DNA). Transformation occurs most commonly in bacteria, both naturally and artificially, and refers to DNA taken up from the environment through their cell wall. CalciumChloridetransformation Calcium chloride transformation is a method of promoting competence. Chilling cells in the presence of divalent cations such as Ca2+ (in CaCl2) prepares the cell membrane to become permeable to plasmid DNA Cells are incubated on ice with the DNA and then briefly heat shocked (e.g. 42 C for 3090 seconds), which causes the plasmid DNA to enter the cell Tipsforefficienttransformationusingchemicalcompetentcells CompetentcellsarebestthawedoniceandDNAshouldbeaddedassoonaslastbitoficeinthetubedisappears IncubateDNAwithcompetentcellsfor30minbeforeheatshock.Expect2foldlossintransformationefficiencyforevery10 minyoushortenthisstep OutgrowthmediumshouldbeSOC,itgives2foldhigherefficiencythenLBmediumandincubationwithshakingincreases theefficiencyto2foldwhencomparedtoincubationwithoutshaking

Electroporation Electroporation is another way to make holes in bacterial (and other) cells, by briefly shocking them with an electric field of 1020kV/cm Plasmid DNA can enter the cell through these holes. Natural membranerepair mechanisms will rapidly close these holes after the shock. . This method is amenable to use with large plasmid DNA.

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Troubleshooting: CloningTherecouldbeseveralfactorsforunsuccessful cloning.Thismakesitimperativetosetup controlsateachstep: 1. Amplification +Ctrl:Anyworkingprimersetwith sameDNA Ctrl:H2OinsteadofDNA 2. Restriction: +Ctrl:Knownsegmentwithsame sites Ctrl:H2Oinsteadofwater 3. Ligation: +Ctrl:Mostoftheligationkitshave Cutvectorascontrol.Alternatively Lambda/HindIIIsamplecantbe used. 4. Transformation: +Ctrl:UncutVector(Circular) Ctrl:CutVector(Linear)

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EXCEPTION TO THE RULE OF THUMBDoesincreasingTa improvesspecificityofprimeralways?No,notalways It is well known that with increase in annealing temperature for PCR favors highly specific binding between primers at template of maximum complementarities. But, this may not be true with every primer set. In the gel snap (Gradient PCR) below with increase in Ta, specificity increases in primer1 but it decreases in case of primer 2 and 3. Nonspecific products were formed, those formed at lower temperatures were presumably due to annealing of primers to nonspecific sites on the template. It is unclear why nonspecific products were formed at temperatures higher than the Ta0PT, but this was a consistent finding from Rhychlik et. Al (1990).

L|Primer1|L| Primer2||Primer3|1kb5556.55859.16162.31kb5556.55859.16162.35556.55859.16162.3

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A question from last seminarCanwereamplifyaPCRproduct?Yes,wecan1. 2. 3. 4. Useproofreadingenzymeinalltheamplificationstoavoidmutationsanderrorsespeciallyatprimer bindingsites. CleanamplifiedDNAeitherbyPCRpurificationkitorGelelutionmethodbeforeusingfornextroundof amplification Diluteamplifiedproductbeforereamplificationtobeusedastemplate. Designprimersinternaltosequenceamplifiedinfirstcycle.

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END OF PRESENTATION

THANK YOU FOR YOUR ALACRITY AND PRECIOUS TIME

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An Abandoned Presentation for Sure Shot Cloning Atul Kakrana