Recombinant DNA = DNA in which genes from two different sources (often different species) are combined in vitro into the same molecule Central to genetic engineering Does not occur in nature (not related to bacterial plasmids entering chromosomes)
Biotechnologymanipulation ofbiological organisms(usually with DNAitself)
DNA IsolationBefore DNA can bemanipulated, it needs tobe isolated from thecells.
DNA IsolationSteps:1. Disrupt cellmembrane with adetergent2.Precipitate DNAwith ethanol3.Retrieveprecipitated DNAand storehttp://www.cambio.co.uk/library/images/html_images/masterpure_complete_graph.gifXResuspendDNAStep 1Step 2Step 3
DNA Amplification In vitro: PCRPolymerase chain reactionPurpose: to increase the amount of DNADNA amplificationCreate millions of copies of a specific DNA sequencesyntheticallyDNA replication in a tube
BrainstormWhat are the major steps in DNA replication?What substances do you need in each step of DNAreplication?
Comparing DNA ReplicationSteps Natural PCRUnwindingOri on DNA templateHelicaseDNA templateHeatPrimingRNA primerPrimaseDNA primer (2)Annealing temperatureElongationNucleotidesDNAPNucleotidesTaq polymeraseTerminationEnd of chromosomeMeet anotherreplication bubbleEnd of DNA templateChange in temperature
PCR Animationhttp://highered.mcgraw-hill.com/olc/dl/120078/micro15.swfhttp://www.dnalc.org/resources/3d/19-polymerase-chain-reaction.html (1: 27)Show types of fragments created and counts theirnumbers (2:53)http://www.youtube.com/watch?v=eEcy9k_KsDI
PCRAt which cycle do you get thecorrectly sized target sequence?http://enfo.agt.bme.hu/drupal/sites/default/files/pcr_0.png
Primerssynthetic sequencesingle-stranded DNA (20-30 nucleotides)provides the needed 3’OH for polymerization2 primers used complementary to each end of thetarget region of the DNA
What’s up with Taq?Why can’t we use the DNAP that is found in mostbacteria?
Taq PolymeraseDNAP Isolated from bacteria(Thermus aquaticus) that live inhot springsHeat stable enzyme (canwithstand extremetemperatures)http://www.amath.washington.edu/~qian/TheScientist/TheScientists_files/geyser.jpg
Water baths to thermal cyclerBefore taq3 water baths at 3temperaturesre-addition of DNAP atthe beginning of eachsynthesis stephttp://www.molecularstation.com/molecular-biology-images/data/509/old-pcr-machine.JPG
PCR EfficiencyThermal cycler:has a plate thatheats and coolsProcess takes 1–2hoursAfter 30 cycles, 230(more than abillion) copies ofDNA can beproduced.http://biolot.com/upload/12_1.jpg
Kary MullisDeveloped PCR in 1986Received Nobel Prize in 1993Interviews Naming PCRhttp://www.dnalc.org/view/15138-Naming-PCR.html Making many DNA copieshttp://www.dnalc.org/view/15140-Making-many-DNA-copies-Kary-Mullis.html Finding DNA to copyhttp://www.dnalc.org/view/15139-Finding-DNA-to-copy-Kary-Mullis.html
Procedure and RationaleTutorialshttp://www.sumanasinc.com/webcontent/animations/content/pcr.htmlhttp://www.dnalc.org/ddnalc/resources/pcr.htmlPCR Virtual Labhttp://learn.genetics.utah.edu/content/labs/pcr/Game & lecturehttp://nobelprize.org/educational_games/chemistry/pcr/game/index.htmlAbout 20 minutes long (with lecture)
DNA Amplification in vivoBacterial cells are transformed (uptake externalDNA)Bacteria are grown in a liquid mediumAs the total number of bacterial cells increase thetotal amount of DNA will increasehttp://www.viewingspace.com/genetics_culture/pages_genetics_culture/gc_w03/kac_webarchive/genesis_page/transformation/trans.jpg
Methods of BacterialTransformationtreat bacteria to make cell walls permeable touptake genetic materialCold CaCl2 treatment followed by heat-shockingmakes cell membrane leaky and more permeableElectroporationelectric current to increase cell permeability
in vitro and in vivo DNAAmplificationSystem in vitro in vivoDefinitionDNAamplificationAdvantage ofsystem
in vitro and in vivo DNAAmplificationSystem in vitro in vivoDefinition Outside of naturalenvironmentInside natural environment(cellular)DNAamplificationAdvantage ofsystem
in vitro and in vivo DNAAmplificationSystem in vitro in vivoDefinition Outside of naturalenvironmentInside natural environment(cellular)DNAamplificationPCR Bacterial cell transformedwith DNAAdvantage ofsystem
in vitro and in vivo DNAAmplificationSystem in vitro in vivoDefinition Outside of naturalenvironmentInside natural environment(cellular)DNAamplificationPCR Bacterial cell transformedwith DNAAdvantage ofsystemDNA can be in apartially degradedstate and still beamplifiedfew errors in replicationdue to proofreading [thuslimitation to the number of timesPCR cycle can be repeated]
Restriction EnzymesBiological “scissors”Endonuclease: breakphosphodiester bondswithin a nucleotide chain(as opposed to at theends of a chain)Fig. 20.2
Restriction Enzyme FunctionFound naturally in bacteria“immune system” of bacteriaprotect bacteria against intruding DNA from otherorganisms (phages, other bacteria)recognize short nucleotide sequences in the foreignDNAcut covalent phosphodiester bonds of both strandsof DNA, rendering foreign DNA harmless
Restriction SiteEach restriction enzyme has a specific sequencethat it recognizes and a specific location on thesequence where it cutsRestriction site: sequence recognized and cut byrestriction enzymeCharacteristic of restriction site:4-8 bp in lengthPalindromic: same sequence on complementarystrand in opposite orientationFig. 20.25’ G A A T T C 3’3’ C T T A A G 5’
Restriction Enzyme Digestion Enzyme cuts at thesame site on both strands Restriction fragments:pieces of DNA created byrestriction enzymes Resulting fragment can have: Sticky End: a single-stranded end of the restriction fragment Blunt ends: straight ends without any single-stranded regionsFig. 20.2
Example: Sticky end with 5’overhang5’ overhang with EcoRI digestion5’ G A A T T C 3’3’ C T T A A G 5’5’ G 3’ 5’ A A T T C 3’3’ C T T A A 5’ 3’ G 5’
Example: Sticky end with 3’overhang3’ overhang with PstI digestion5’ C T G C A G 3’3’ G A C G T C 5’5’ C T G C A 3’ 5’ G 3’3’ G 5’ 3’ A C G T C 5’
Example: Blunt endblunt ends with SmaI digestion5’ C C C G G G 3’3’ G G G C C C 5’5’ C C C 3’ 5’ G G G 3’3’ G G G 5’ 3’ C C C 5’
Naming Restriction EnzymesRestriction enzymes are named according to theorganism from which it was identified.Example: EcoRIE - Escherichiaco - coliR - strain RY13I - 1st enzyme in this strain
Recombinant DNA DNA in which genes from two different sources (oftendifferent species) are combined into one molecule Usually the gene of interest is inserted into a bacterialplasmid Why do you think a bacterial plasmid is used?
Fig. 20.3Forming Recombinant DNA Restriction enzyme digestion of plasmid and gene ofinterest Hybridization of matching sticky ends on gene ofinterest and plasmid DNA ligase seals gene of interest with plasmid
Activity: Recombinant DNAYou are given either a gene of interest (linear) or aplasmid (circular)Cut out your DNADigest it with the given restriction enzymeFind the person with a matching sticky end toform the recombinantAdditional: Research the data pair. Prepare ashort (3-4 sentence) write-up that relates to thetwo terms.
Gel ElectrophoresisSize separation of nucleic acids by moving themthrough a gel medium using electric currentCan also be used for protein
Gel ElectrophoresisDNA is negatively charge (phosphates) thus willmove toward the positive electrode
Making the gelLiquid solutions of thegel medium is pouredinto a mould andallowed to set andsolidifyNotice the comb isplaced at one end of thegel. When it is removedit creates the wellswhere DNA is loaded.
Positive (+)electrodeNegative (-)electrodeWhere DNAis loadedonto gelBlue liquidis bufferhttp://cg.scs.carleton.ca/~morin/teaching/compbio/electro.htmlHorizontal Gel
Other MaterialsLiquid buffer containing ions:Provides a medium for the flow of electric currentPrevents gel from overheating and drying out.Coloured dyes mixed with DNA:track distance travelled on the gel by DNAIncrease density of DNA so that it sinks to bottom ofwells