Biology11ALab#4 (BringyourTexttoLab)
DNAExtraction,StudyoftheDNAModelandMitosis
Pre-LabQuestions(Findtheanswersinthebook)Answerincomplete
sentences.
1.Whatismeantbytheanaphasestageofmitosis?
2.WhichscientistsdescribedthestructureofDNA? Whatwastheyear?
3.WhatarethesidesoftheDNAladdermadeof?
4.Whatarethe"rungs"oftheDNAladdermadeof?
5.DNAcanbefoundinwhattwoorganelleswithintheanimalcell?
I n t r o d u c t io n
Thegenetic,hereditarymaterial,DNA,isthelanguageoflife.DNAdetermineswhetheran
individualwillbeadogorahuman,anditunderliesphysicalandbehavioralcharacteristics.Itis
foundinthenucleusofeachcell,coiledinlongpackage-likestructurescalledchromosomes.
Uncoiledtherewouldbeabout6feetofDNAineachcellinyourbody.However,itisaverythin
thread,onlyafewnanometerswide,andisimpossibleforustoseedirectly.
YouhaveheardofthescientistswhofirstdescribedDNAin1953 –JamesWatsonandFrancis
Crick,withthehelpofX-raydiffractionimagestakenbyRosalindFranklin.Thediscoveryofthe
structureofDNArevolutionizedthestudyofbiology.Duringthe1950’sand60’sscientists
discoveredthatchromosomesarecomposedofgenes, sectionsofDNAthatcodeforaparticular
protein. Further,researchersfiguredouthowthemessagecontainedinDNAisusedbycellsto
buildproteins.Proteinsarenecessaryforlifebecausetheycontroleverycellularprocessandthey
providebuildingblocksforourtissuesandorgans—youareliterallybuiltonascaffoldingof
proteinssuchascollagen.
Inthe60yearssincethedescriptionofthedoublehelix,therehavebeenastounding
discoveriesconcerningthefunctionsofDNA.Inthe21stcentury,thehuman genome (thelibrary
ofhumangenes)hasbeenmapped.Humangenomeresearchhasrevealedthatthereare30,000to
40,000genes.Theseareinheritableinstructionsforhowtobuildahumanbeing.Inaddition,the
genomesofmanyotherspecieshavealsobeenmapped,providingpowerfultoolsforcomparative
studies.Theinformationisbeingusedtobetterunderstandhealthanddisease,includingthe
relativerolesofheredityandenvironmentinthedevelopmentofcomplexdiseasessuchas
diabetesandcancer.Inthefuture,thestudyofDNAandproteinsmayprovidetheabilitytotailor
drugsspecificallysothatindividualscanpreventandfightdiseasemoresuccessfully.
1
DNAE xt raction
AlllivingcellscontainDNA.TheDNAinacellisabout100,000timesaslongasthecell
itself,yetonlytakesuponlyabout10%ofthecell’svolume.TheDNAmoleculesare
foldedandpackedintochromosomesinthenucleusofacell.BeforeDNAcanbestudied,
itmustbeextractedfromcells.Today,wewillextractDNAfromplantcellsusinga
strawberry.
ThereareafewbasicstepsinDNAextraction.Thecellmustbelysed(brokenopen)to
releasethenucleus.Plantcellshaveacellwallinadditiontothecellmembrane.Thecell
walliscomposedofcellulose,apolysaccharidecomposedofchainsofglucosemolecules.It
formsatoughbarrierthatmustbebrokendowntoallowthecontentsofthecelltogetout.
Thefrozenstrawberriesaremashedinabaggietobreakthecellulosemolecules.
Otherthanthecellwall,plantshaveacellmembranecomposedprimarilyoflipidsthat
musttoobebroken.ThenucleusifpresentmustalsobeopenedtoreleasetheDNA.
Detergentsbreakdownthelipidmembranesthatenclosetheentirecellandthenucleus
inamannersimilartowashingagreasypan.ThefragmentsofDNAhaveanegative
electricalcharge.Thatnegativechargeisneutralizedbythepositivesodiumionsin
solution.ThisneutralizationofthenegativechargesonDNAallowsittoprecipitatein
alcohol.Withoutthesalt,theDNAremainsnegativelychargedandwillstayintheaqueous
partofthesolution.OncetheDNAisreleasedintosolution,itwillbeprecipitatedbythe
ethanolalcohol.Precipitationistheactofmakingmoleculessticktogether.Inthe
presenceofethanol,DNAisnolongersolubleinwaterandseparatesoutofthesolutionso
thatwecanisolateandlookatit.Ethanolworksbypromotingionicbondsbetweenthe
Na+(fromthesalt)andthePO3-(fromtheDNAbackbone).
Today’sObjectives:
1.ExtractDNAfromaoncelivingthing,astrawberry.
2.UnderstandthestructureofDNAbylookingatamodel
3.UnderstandthebondingstructureofDNAbyusingpuzzlepieces
Part1ProcedureforStrawberryDNAExtraction
Materialsneededpergroupoftwo.Pleaseworkinpairs.
1frozenstrawberry 125mlofwater
1baggie
1coffeefilter
10mlofdetergent 2cupsorbeakers
2.5mlofsalt Approximately100mlofalcohol
1glasspipette
1.Placeonefrozenstrawberryinyourbaggie,sealitupandgentlymushitupuntilitturns
2
intostrawberrypuree.
2.Useacuporbeakertomixthedetergent,saltandwatertogether.
3.Pourthesaltmixtureintothebaggieandsealthebaggieagain.
4.Oncethemixturelookswellblended,filteritthroughacoffeefilterintoyourclean
beaker.
5.AidthefilteringprocessbyGENTLYsqueezingmoreofthemixtureintothebeaker.
6.Precipitation:Evaluatethevolumeofyour
strawberry“smoothie”andaddanequalamountof
thealcoholtothebeaker.
7.WatchtheDNAprecipitateoutofthesolution(give
5minutes).TheDNAwillfloatinthealcohol.Youwill
seethreadsthateasilyspool.Takeapictureofitfor
yourreport!
it
ForPart1ofLabWrite-upyouwillneedtoinclude:
•
•
DescribetheamountofDNAyouextracted,whatitlookslikeetc.Aphotoworksif
youtakeone!
Answerthequestionsthatfollow.Numberthequestionsandtype(doublespaced)
incompletesentencessothatevenwithoutthequestionpresentthesentenceis
understood.
Questions(Answerincompletesentencesalways.Numberthem.)
1.WhatwasthepurposeofusingdetergentintheextractionofDNA?
2.WhatwasthepurposeofusingsaltintheextractionofDNA?
3.WhatwasthepurposeofusingalcoholintheextractionofDNA?
4.WhatstructureareyoulookingatwhenyouseeDNAasablobofJello?
5.WoulditbepossibletoextractDNAfromanimalcellsusingthemethodyouusedforaplant?
Whyorwhynot?
6.“Apersoncannotseeasinglecottonthreadfourclassroomsaway.Butifyouwoundthousands
ofthreadstogetherintoarope,itwouldbevisibleatthesamedistance.”Isthisagoodanalogyfor
our“seeing”ofDNA?Explain.
3
SomeInterestingFactsabouttheCFTRproteinsequenceofCysticFibrosisthatwe
spokeofinlecture.
•
•
•
•
•
•
TheCFTRgeneisfoundonchromosome7
Itcontains471,745nucleotides,codesforaproteincontaining1480aminoacids.
Thereareapproximately900differenttypesofmutations,someofwhichmayresultin
milderformsofthedisease.
Approximately1in31Americansarecarriersofthecysticfibrosisgene.
CFTRarefoundinthepancreas,lungs,colonandreproductivesystem
Inmanycysticfibrosiscases,thedefectiveproteinismadeinthecells,butneverreaches
thecellmembrane.Processingofthefaultyproteinisstoppedwhenthecell’squality
controlmechanismsrecognizethattheproteinisfoldedincorrectly.Insteadoftaggingthe
proteinfortransporttothemembrane,thedefectiveCFTRismarkedfordegradation.
Part2DNAModel
LookatthemodelofDNAondisplay.
1.Theyellowballswithtworedonesattachedrepresent______________________.Drawtheshapeof
thismolecule(findthisinyourtext).
2.Thesugarisrepresentedbywhatcolorballs?Drawtheshapeofthesugar.
3.ThesearethenitrogenousbasesfoundinDNA.GuanineandCytosinearepaired,whereas
AdeninepairswithThymine.
PURINES
Adenine
Guanine
PYRIMIDINES
Cytosine
Thymine
Howthepairing
happensbetweenthenitrogenousbases:
4
Findthebasepairingseenheretotheleft,
onthemodel.
3.Writedownthesequenceatwhichthesepairsoccuronthemodelfromtheverybottomrungof
theladderuptothe10thstep.(Thereare16altogether)NoticewhetheryouarereferringtoA-Tor
T-A.AlsonoticeifyouhaveC-G,orG-C.Howcanyoutell?
1._______________________ 6.______________________________
2._______________________ 7._____________________________
3._______________________ 8._______________________________
4._______________________ 9.______________________________
5._______________________ 10.____________________________
Part3DNAPuzzle
5
Obtainalargebaggiethatcontainsthepuzzlepieces.Thekitshouldcontaineverything
youneedtoassembleasectionofDNA.Selectoutthefollowingpieces:
Phosphates (yellow)
Deoxyribose (darkpink)
Guanine
(darkblue)
Alanine
(lightgreen)
Thymine
(lightblue)
Citosine
(darkergreen)
WewillnotbeusingthelightpinkriboseorwhiteUracil.Usetheindicatedpiecestoform
adoublestrandedmoleculeofDNAinwhichtheleftsideoftheladderfollowsthisorderof
tennucleotides:
A-G-C-A-T-T-G-C-C-G
Youwillneedtousethephosphatesanddeoxyribosetoformthesidesoftheladder.
Drawtwoentirenucleotidesincludingallthedetailyoucan.
Part4OnionRoottips(Mitosis)
Getamicroscopeandaslideofanonionroottip.Findcellsthatareundergoingmitosis.
Findacellinprophase,metaphase,anaphase,telophaseandcytokinesis.Makea
DETAILEDdrawingofeachofthesestagesonaseparatepieceofnon-linedpaper.Hand
theseinwithyourreport.Pleasemakethedrawingdetailed.
WRITINGUPTHISLAB(Alwaystyped,doublespaced).Duenextlabperiod.
Part1AttachadescriptionofyourDNA“blob”.Answerquestions1-6.
Part2 DNAModelObservation.Listtheorderofthenucleotidesfrom1-10.
Part3DNAPuzzle.Drawtwonucleotidesplusthephosphateanddeoxyribose.i.e.draw
onefullstepoftheladderwithitsonerungandtwosides.Itneedsdetail.(Don’tjusttrace
aroundonepuzzlepiece).Showthebondsandtheelements.
Part4OnionRootTip.Drawthe5stagesofcelldivisiononnon-linedpaperasseenunder
yourscope.Youwillnotseeasmuchdetailasthedrawingsinyourtext.Makeitclear.Use
yourtexttobecertainthatyouarelookingatthecorrectthing,butpleasedonotcopy
thosedrawings.Labelclearly.
6