BCH5413 Exam 1 Questions And Answers

BCH5413 Exam 1 Questions And
Answers
-tCORRECTttANSWER-
TotdeterminetiftatspecifictproteintinteractstwithtatspecifictDNAtsequencetintthetnucleustoftatce
lltorttissue.

5.tWhattdoestthetMeselsontandtStahltexperimenttconfirm?t-tCORRECTttANSWER-
Int1958tMeselsontandtStahltpublishedttheirtexperimenttoftDNAtreplicationtintEscherichiatcoli
.tIntthistexperimentttheytstartedttheirtDNAtmoleculetwiththeavytnitrogentbasestandtwhentthe
ytaretseparatedtthetnewtstrandtmadetwastwithtlighttnitrogentbasestandtthetnewtdaughtertstra
ndteachtwastfoundttothavetonetheavytandtonetlighttstrandtwhichtprovedtthetDNAtreplicationt
mechanismtastsemiconservativetDNAtreplicationtmodeltastproposedtbytWatsontandtCrick.

Aftertanyttypetoftmutagenesististcomplete,tthetadditionaltstepstofttransformation,tselection,t
miniprep,tandtsequencingtaretneeded.tExplaintwhyteachtistnecessaryttotcompletetthetmuta
genesistproject.t-tCORRECTttANSWER-
Transformation:tBacteriatuptaketgenetictmaterialttotgaintatfunction,tplatetthetbacteriatontme
diatthattcontainstantibiotic,tcoloniestthattgrowthavetthetplasmid
Selection:tPicktatcolonytandtgrowtthemtintliquidtLBt(+tselection)tovernightttotgrowtmoretoftthe
tbacteria

Miniprep:tUsedttotisolatetthetplasmid
Sequencing:tIntthiststeptwetaretscreeningtfortthetmutationtintorderttotchecktthattthetmutationt
actuallyttookteffect,tusuallytthoughtsangertsequencingtandtusetatchromatogram,twantthights
ignaltstrengthtfortthetnewtmutation

Aftertisolatingtintacttchromatintfromtcells,tlightlytdigestingtittwithtmicrococcaltnucleasetandtr
unningtittontantagarosetgel,tyoutseetatbandingtpatterntoftrepeatingt200bptfragmentst(200,t40
0,t600tetc).tExplaintthistresult.t-tCORRECTttANSWER-
Thet200bptbandingtpatterntistshowingtthattthetmicrococcaltnucleasethastcuttbetweenteachti
ndividualtnucleosome.tThet400bptpatterntistshowingtthattthetnucleasethastcuttbetweentever
yt2tnucleosomestandtthet600bptpatterntisteveryt3tnucleosomes,tetc.tThistexperimenttwastpe
rformedttotshowtthattpartstoftDNAtaretprotectedtfromtthetnucleasetbytnucleosomes.tWhentth
etconcentrationtoftnucleustincreases,ttheretaretmoretindividualtnucleosomes.
Nucleasestonetaret200bpteach
200bptfragments=tindividualtnucleosome

AftertonetcycletoftPCR,thowtmanytDNAtfragmentsttotyouthavetthattexactlytmatchtyourttargett
sequence?tExplaintyourtanswer.t-tCORRECTttANSWER-

,WhentPCRtiststartedtwithtonetdsDNAtfragment,ttheretwilltbettwotexacttcopiestoftthettargettse
quencetaftertonetcycle.
Forteachtcycle,tthetPCRtdoublestthetexistingtnumbertoftcopiestoftDNAtsequencet=tlogarithm
ictamplification.

Allelet-tCORRECTttANSWER-
onetofttwotortmoretversionstoftDNAtsequencet(atsingletbasetortatsegmenttoftbases)tattatgiven
tgenomictlocation.tAntindividualtinheritsttwotalleles,tonetfromteachtparent,tfortanytgiventgeno

mictlocationtwheretsuchtvariationtexists.

BaittandtPreytMethodttotstudytprotein-proteintinteractionst-tCORRECTttANSWER-
Totstudytprotein-
proteintinteractionstyoutcantusetthet"baittandtprey"tmethodtwithtfusiontproteins.tFirsttyouthav
ettotimmobilizetthetfirstt"bait"tproteinttotagarosetbeadstthentwashtawaytanytunboundtprotein.t
Thentyouthavettotaddtyourt"prey"tproteintandtlettthemtincubatetandtinteracttbeforetwashingtt
hetunboundtproteinstatsecondttime.tYoutcantthentelutetthetproteinstthatthavetinteractedtfrom
tthetagarosetbeadstandtruntantSDS-

PAGEtgelttotvisualizetthem.tThetmosttcommontmethodtfortvisualizingtthesetinteractionstwo
uldtbetatwesterntblot.tFromtthetgeltyoutcantseetthetbandstoftinteractingtproteinstandtboundt"b
ait"tproteinst.

ChIP:
HowtistthetDNAtthattistboundttotthetproteintoftinteresttisolated?t-tCORRECTttANSWER-
addtantibodytoftinteresttandtperformtimmunoprecipitationttotpulltthetantibodytouttwithtthetDN
Atoftinteresttboundttotit

ChIP:
HowtwouldtantinvestigatortdeterminetiftatspecifictDNAtsequencetistboundttotthetproteintoftint
erest?t-tCORRECTttANSWER-PCRtusingtgene-
specifictprimersttotdetecttDNAtoftinterest,tpositivetproducttconfirmstproteintpulledtwastboun
dttotDNAtsequencetoftinterest

ThetproteintisolatetfromtthetimmunoprecipitationtassaytistdegradedtandtPCRtistperformedtu
singtprimerstspecificttotthetDNAtsequencetoftinterest.tThetPCRtproducttistthentevaluatedtusi
ngtagarosetgeltelectrophoresis.tAtpositivetPCRtproducttsuggesttthattthetisolatedtproteintwas
tboundttotthetDNAtsequencetoftinterest.




ChoosingtantE.tcolitproteintexpressiontsystemthastatlottoftadvantagestsuchtastyield,tcosttand
tease.tDiscusst(nottjusttlist)ttwotreasonstwhytatresearchertmighttnottchoosetthisttypetoftsyste

m.tBetspecific,twhattmightttheirtgoaltbetandtwhytwouldn'ttthistsystemtworktfortthem?t-
tCORRECTttANSWER--

AntE.tcolitproteintexpressiontsystemtmighttnottbetsophisticatedtenoughtfortthetproteintwetare
ttryingttotstudy,tandttheretcouldtbetdownstreamteffectstontthetprotein'stexpression.t




AntexampletDr.tGumztgavetwastthetlimitationtthattE.tcolitsystemstcannottsupporttpost-
translationaltmodifications-

, sotiftourtproteintoftinteresttneedsttotbetmethylatedtortphosphorylated,tantE.tcolitsystemtistnott
t

atgoodtmatchtfortwhattwetarettryingttotstudy.-
AtsecondtlimitationtistthatttheretaretcodontusagetdifferencestbetweentE.tcolitcellstandtmamm
aliantcells.tWetmighttencountertwhattDr.tGumztdescribedtast"experimentaltdifficulty"twhenttr
yingttotapplytfindingstfromtantE.tcolitproteintexpressiontsystemttotatmammaliantproteintexpre
ssiontsystem,tsincetproteintexpressiontmaytnottalwaystbetthetsame

Comparetandtcontrasttthetmethodtoftbasetdetectiontbetweentpyrosequencingtandtionttorren
ttsequencing.t-tCORRECTttANSWER-
Thetbasetdetectiontmethodstoftpyrosequencingtandtionttorrenttsequencingtdetecttonetnucle
otidetattattime.tAdditionally,tthroughtthetbreakingtoftthetphosphatetbonds,tthetmethodstaretin
directtprocessesttotdeterminetthetnucleotidetpresent.
Fortpyrosequencing,tiftthetnucleotidetthattistflowedtacrosstthetplatetistincorporatedtastthetne
xttnucleotide,tthentpyrophosphatet(PPi)tistreleased.tThen,tthetPPitistbrokentaparttwithtsulfur
ylasettotproducetATP.tThetATPtistutilizedtbytluciferasettotproducetlight.tThetpyrosequencingt
methodtdetectstPPi.
Fortionttorrenttsequencing,tiftthetnucleotidetthattistflowedtacrosstthetplatetistincorporatedtastt
hetnexttnucleotide,tthentHydrogentiontistreleasedtwithtthetbreakagetoftthetphosphatetbonds.t
ThetHydrogentiontistthentutilizedttotdetecttatchangetintpH.tThetionttorrenttsequencingtmetho
dtdetectstHydrogentions.

CoretHistonest-tCORRECTttANSWER-H3,tH4,tH2A,tH2B

DescribethowtPCRtcantbetmodifiedttotmaketittquantitative.t-tCORRECTttANSWER-
UsingtfluorescencetacrosstPCRtcycles.tAstPCRtcyclestprogress,tthethighertthetfluorescentt
signaltis,tindicatingthighertmRNAtexpressiontlevels.
TaqMantProbe-
BasedtAssay:tusestatprobetwithtatreportertandtquencher.tReportertwilltonlytfluorescetwhentd
islodgedtfromtthetprobetandtawaytfromtthetquencher.tDislodgingtoccurstduringtastDNAtpoltm
ovestthroughtthetprobetduringtamplification.
SYBRtGreentItDyetAssay:tthistfluorescenttbindstindiscriminatelyttotdsDNA;thence,tastamplifi
cationtproceedstandtmoretDNAtcopiestaretmade,tthetfluorescenttsignaltincreases.

Describethowttransfectiontoftplasmidstintotmammaliantcellstistdifferenttfromttransformationt
oftplasmidstintotbacterialtcells.t-tCORRECTttANSWER-
Intmammaliantcells,ttransfectiontistlipid-
mediatedtintwhichtDNAtistincubatedtwithtlipofectiontreagentttotformtatDNAtlipidtcomplex

DescribetsimilaritiestbetweentDNAtandtRNA.t-tCORRECTttANSWER-
1.tBothtDNAtandtRNAtaretlargetbiologicaltpolymers
2.tBothtDNAtandtRNAtaretnucleictacids
3.BothtDNAtandtRNAtconsiststoftsugar,tnitrogenoustbases,tandtatphosphatetbackbone
4.tOntbothtmoleculestGuaninetpairstwithtCytosine
5.tBothtstoretgenetictinformation