30/1/2017 DNAANALYSISsimulatingrecombination

Name(s):_________________________________________________Date:______

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?

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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
______________________________________

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