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TOOLSOFBIOTECHNOLOGY
HLeeYuJsuicoJunsay
DepartmentofChemistry
SchoolofScienceandEngineering
AteneodeManilaUniversity1
BiotechnologyistheapplicaAonofbiologicalmaCerforusefuloperaAons.
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BiotechnologyistheapplicaAonofbiologicalmaCerforusefuloperaAons.
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1. HowdoyousequenceDNA?2. HowdoyouvisualizeDNA?3. HowdoyoumakemorecopiesofDNA?4. Withalltheseinmind,canyouchange
anorganism’sgenes?
DNASEQUENCINGANDVISUALIZATION
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DNAcanbecharacterizedbysizebymeansofelectrophoresis.
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SIZEM
ARK
ERS
+++ +++++++++
AgaroseGelElectrophoresis(AGE)isthemostcommontechniqueforDNAseparaAonandsizedeterminaAon.
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DNAisvisualizedbymeansofUV‐acAveagentsthatbindtoDNA.ThiscanbeviewedunderUVlight.
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DNAsequencescanbeprobedforspecificsequencesthrublo_ngmethods(SouthernBloCngbyEdwinSouthern)
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DNAsequencescanbeprobedforspecificsequencesthrublo_ngmethods(SouthernBloCng)
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• Probe– AlabeledsinglestrandofDNAorRNA
– seekoutacomplementarysequenceofsingle‐strandedDNA
– Usedinscreening
• Primer
– Singlestranded– RequiredbyDNAPolymerasetosynthesizeDNA
DNAsequencescanbeprobedforspecificsequencesthrublo_ngmethods(SouthernBloCng)
10SOUTHERNBLOT=DNA
NORTHERNBLOT=RNAWESTERN
BLO
T=PR
OTEIN
EASTERN
BLOT=?!?
TheSangermethodmakesuseofaDNAyouwanttosequence,ashortprimer,DNAPolymerase,dNTPsandspecificddNTPs.
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TheSangermethodmakesuseofaDNAyouwanttosequence,ashortprimer,DNAPolymerase,dNTPsandspecificddNTPs.
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TheSangermethodmakesuseofaDNAyouwanttosequence,ashortprimer,DNAPolymerase,dNTPsandspecificddNTPs.
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TheSangermethodmakesuseofaDNAyouwanttosequence,ashortprimer,DNAPolymerase,dNTPsandspecificddNTPs.
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DNAAMPLIFICATION:POLYMERASECHAINREACTION
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ArapidandversaAleinvitromethodtoamplifydefinedtargetDNAwithinaheterogeneouscollecAonofDNAsequences(genomicDNAorcDNA)wasinventedbyKaryMullisin1984.HecalleditpolymerasechainreacSon
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5’5’3’
3’DNAfragment(blue)withsequenceofinterest(orange)
FreedNTPsTaqPolymerase
sense
anAsense
Primers
5’
3’5’
3’
Figure1.DissolveallyourcomponentsinabuffersoluAonandplacetheminPCRTubes.LoadPCRtubesinPCRmachinehCp://www.molecularstaAon.com/molecular‐biology‐images/data//509/pcr‐tubes1.pnghCp://www.molecularstaAon.com/molecular‐biology‐images/data/509/pcr‐machine.jpg
Figure2.PCRcyclesthrough3temperaturesteps.ThefirstofwhichistheDenaturingStep:Holdat94Cfor1minutetoseparatethedoublestrandedDNAtoitsindividualstrands.
5’
5’3’
3’sense
anAsense
Figure3.ThesecondstepistheAnnealingstep:Loweringtemperatureto50‐65CdependingonthemelAngtemperatureoftheprimers,holdingforabout1minute.ThisstepallowsfortheaCachmentoftheprimerstosequenceofinterest.
5’
5’3’
3’sense
anAsense
5’
3’5’
3’
Figure4.ThethirdstepistheExtendingstep:increasetemperaturetoaround75‐80C(orwherevertheenzymeoperatesatitsopAmum).HoldforavariableAmedependingonthelengthofsequencetobeamplified.Itusuallytakesapolymerase1000bpperminute.Atthisstep,freedNTPsareusedbythepolymerasetomakecopiesoftheDNAofinterest.
5’
5’3’
3’sense
anAsense
5’
3’5’
3’
5’
5’3’
3’sense
anAsense
5’
5’
Figure5.ThisshowsthetemperatureprofileofthePCRprocesswiththecorrespondingstepsinvolved.
hCp://www.mun.ca/biology/scarr/PCR_sketch_3.gif
5’
5’3’
3’sense
anAsense
5’5’
3’3’
Figure6.Amer1cycle,youhavenowtwocopiesofyourDNA.Thefirstcyclecreatesacopywithextraneoussequence(indarkgreen).Anothercyclewillcreateafragmentthatonlycontainsyoursequenceofinterest(orange),andthiswillbefurtheramplifiedtocreatefragmentsofyourdesiredlength.
sensecopy
anAsensecopy
5’
5’3’
3’sense
anAsense
5’
5’
3’
3’sensecopy
anAsensecopy
5’ 3’sensecopy2
5’3’ anAsensecopy2
5’3’ anAsensecopy2
5’ 3’sensecopy2
Amercycle2
Figure7.Thisanagarosegelof3AssuesamplesraninPCRwithtwoprimersets.NoAcethatonlyTissue2andTissue3containfragmentofinterestascomparedtoaposiAvecontrol.Onecanalsosee,heavierfragments(encircledinred)whichcouldbethoselongerfragmentswithextraneousDNA.
DNAMolecularWeightLadderPrimerset1 Primerset1 Primerset2Primerset2
hCp://upload.wikimedia.org/wikipedia/en/d/d0/Roland_Gel.JPG
hCp://users.ugent.be/~avierstr/principles/pcrcopies.gif
Figure8.NumberoffragmentsexponenAallygrowunAlaplateauduetolimitaAonsintheamountofcomponents(dNTPs)
hCp://www.ionchannels.org/content/images/14‐02.jpg
RECOMBINANTDNATECHNOLOGY
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TheabilitytochangethegeneAccharacterisAcsoffundamentallifeformsisgenerallycalledrecombinantDNAtechnologyorgeneScengineering.
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MakingrecombinantDNAconsistofthecovalentinserAonofaDNAfragmentfromonetypeofcellororganismintothereplicaAngDNAofanothertypeofcell.
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MakingrecombinantDNAconsistofthecovalentinserAonofaDNAfragmentfromonetypeofcellororganismintothereplicaAngDNAofanothertypeofcell.
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ThecarrierofthegeneAcmaterialiscalledavector.RestricSonenzymesareendonucleaseswhichcuttheDNAatspecificpalindromicsites.
MakingrecombinantDNAconsistofthecovalentinserAonofaDNAfragmentfromonetypeofcellororganismintothereplicaAngDNAofanothertypeofcell.
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Twocommonvectorsareincommonuse:PlasmidsandBacteriophage
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Plasmids(1)shouldbeabletoreplicateinarelaxedfashion,(2)shouldbesmall,(3)shouldcontainmarkersforscreeningprogenyand(4)shouldonlyhaveonecleavagesite.
Twocommonvectorsareincommonuse:PlasmidsandBacteriophage
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Bacteriophageorλphageisusuallylarger,canmakemanycopiesoftherecombinantDNAandhaveefficientpackaging
RestricAonenzymescutatspecificsitesandproducesbluntorsAckyends.
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RestricAonenzymescutatspecificsitesandproducesbluntorsAckyends.
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BluntendsarehardtoligatetoDNA,homopolymertailsmayusedinstead.
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RecombinantDNAmustbeintroducedtoacellbymeansofseveralmethods,thenthesecellsmustbegrownandselected.
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RecombinantDNAmustbeintroducedtoacellbymeansofseveralmethods,thenthesecellsmustbegrownandselected.
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RecombinantDNAmustbeintroducedtoacellbymeansofseveralmethods,thenthesecellsmustbegrownandselected.
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RecombinantDNAmustbeintroducedtoacellbymeansofseveralmethods,thenthesecellsmustbegrownandselected.
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Figure 8-39 Molecular Biology of the Cell (© Garland Science 2008)
Figure 8-40 Molecular Biology of the Cell (© Garland Science 2008)
Figure 8-41 Molecular Biology of the Cell (© Garland Science 2008)
