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DNAANALYSISSimulatingRecombination
Materials:Scissors,Tape,StudentHandoutsShowing
Sequences
Instructions
1.CutoutthePlasmidBaseSequencestripsandtapethem
togetherintoonelongstrip.Thelettersshouldallbeinthe
samedirection.Tapethetwoendsofthelongstriptogether
toformacirclewiththelettersfacingout.THISISYOUR
PLASMIDDNA.
2.CutouttheDNABaseSequenceStripsandtapethemtogetherinnumericalorder.ThisisyourHUMAN
DNA,whichcontainsthegeneforinsulinproduction.Thegeneareaisshaded.
3.CutouttheRestrictionEnzymeSequenceCards.Eachcardshowsasequencewhereaparticularrestriction
enzymecutsDNA.
4.Comparethesequenceofbasepairsonanenzymecardwiththesequencesoftheplasmidbasepairs.If
youfindthesamesequenceofpairsonboththeenzymecardandtheplasmidstrip,markthelocationonthe
plasmidwithapencil,andwritetheenzymenumberinthemarkedarea.Repeatthisstepforeachenzyme
card.
Someenzymesequencesmaynothaveacorrespondingsequenceontheplasmid,andthat
someenzymesequencesmayhavemorethanonecorrespondingsequenceontheplasmid.In
thisstep,youaresimulatingtheprocessofchoosingthecorrectrestrictionenzymetorecombine
yourDNA.Withhundredsofrestrictionenzymesavailable,scientistsmustdeterminewhichone
willworkfortheDNAtheywanttorecombine.
5.Onceyouhaveidentifiedallcorrespondingenzymesequencesontheplasmid,identifythoseenzymes
whichcuttheplasmidonceandonlyonce.Discardanyenzymesthatcuttheplasmidintheshadedplasmid
replicationsequence.Youdon'twanttocutoutthisparticulargene,becauseitisnecessaryforthebacteriato
replicateitself.
Whichenzymesfitthiscriteria?______
6.Next,comparetheenzymesyouchoseinstep5againstthecellDNAstrip.Findanyenzymesthatwillmake
twocutsintheDNA,oneabovetheshadedinsulingenesequenceandonebelowtheshadedinsulingene
sequence.MarktheareasontheDNAstripthateachenzymewillcutandmakeanoteofwhichenzymecuts
inthatspot.
7.Selectoneenzymetousetomakethecuts.ThegoalistocuttheDNAstrandascloselyaspossibletothe
insulingenesequencewithoutcuttingintothegenesequence.ExtraDNAmovedtothehostmayhave
unpredictableresults,youareonlytryingtomovetheportionoftheDNAthatcontainsthegeneofinterest.
MakecutsonboththeplasmidandtheDNAstrips.Makethecutsinthestaggeredfashionindicatedbythe
blacklineontheenzymecard.
8.Tapethestickyends(thestaggeredends)oftheplasmidtothestickyendsoftheinsulingenetocreate
theirrecombinantDNA.Inthelab,DNAligaseisusedtobindthestrandstogether.
Congratulations!Youhavesuccessfullycreatedabacterialcellthatcontainsthehumaninsulingene.This
bacteriawillreproduceandcreatemorebacteriawiththegene.Bacteriagrowninculturescannowmass
produceinsulinfordiabetics.
DiscussionQuestions
1.Whywasitimportanttofindanenzymethatwouldcuttheplasmidatonlyonesite?Whatcouldhappenif
theplasmidwerecutatmorethanonesite?
https://www.biologycorner.com/worksheets/DNA_analysis_recombination.html 1/2
30/1/2017 DNAANALYSISsimulatingrecombination
2.Whichrestrictionenzymedidyouuse?_____________Askothergroupswhattheyusedandcomparethe
finaltransgenicplasmids.Whymighttherebesomeofdifferentlengths?
3.Whywasitimportanttodiscardanyenzymesthatcuttheplasmidatthereplicationsite?
4.WhyisitimportanttocuttheplasmidandthehumanDNAwiththesamerestrictionenzyme?
5.Dorestrictionenzymesexistnaturallyinorganisms?Ifso,whatistheirpurpose?
6.Whywouldrestrictionenzymesthatcreated"blunt"endsnotbeasusefulinrecombinationasthosethat
createstickyends?
7.Intheactivity,yousimulatedcreatingarecombinantbacteriaorganism.Foreachofthefollowingmaterials,
indicatewhattheyrepresent?
Scissors_______________________________________Tape
______________________________________
https://www.biologycorner.com/worksheets/DNA_analysis_recombination.html 2/2