BCH Exam 1 Study Guide Questions And Answers

BCH Exam 1 Study Guide Questions And
Answers

-tHowtistthetchromatintsheared?t-tCORRECTttANSWER-
MechanicallytortEnzymatically;tSheartDNAtintotfragments.

2twaysttotincreasetthettransfertefficiencytoftDNAttotatblott-tCORRECTttANSWER-
UsetdilutetHClttotpartiallytdepurinatetthetDNA,twhentsubjectedttotdilutetNaOHtthetDNAtwilltb
etcleavedtattthetsitestoftdepurinationt(thistmakestthetfragmentstsmallertandteasierttottransfer
)

NeutralizetthetpHtoftthetgeltbytsoakingtintatpHt7tbufferedtsolutiont(iftthetpHtisttoothightthetDNA
twilltnottbindttotthetmembrane)




3.tIntthetfiguretbelow,t
a.tWhichtexperimenttistbeingtdone?
b.tWhytisttheretatregiontoftnotbands?
c.tWhatttypetoftinteractiontdoestthistexperimenttdetermine?t-tCORRECTttANSWER-
a.tDNAtfoottprinting
b.tProteintboundttotDNA,tthetproteintblockstthetcleavagetsitestsotnotDNAtwastcut
c.tDNA-proteintinteraction

6.tWheretintthetgenometaretloxPtsitestlocatedtintatCre/Loxtmodel?t
a)tAdjacentttotthetCretlocustexpressedtintatdesiredtcellttypet
b)tOnteithertsidetoftthetgenetthattisttotbetknockedtoutt
c)tRandomtsitestthroughouttthetgenomet
d)tAlltoftthetabovet-tCORRECTttANSWER-
b)tOnteithertsidetoftthetgenetthattisttotbetknockedtout

a.tExpandtCRISPRtCast-tCORRECTttANSWER-
ClusteredtRegularlytInterspacedtShorttPalindromictRepeats)t&tCRISPR-associatedt(Cas)

Explaintlibrarytscreeningtusingtplaquethybridization.t-tCORRECTttANSWER--
tlaytmembranetontpetritdishtandtlifttofftatreplicatoftplaques

-tDNAtontfiltertcorrespondsttotthattoftthetplaques
-tblocktfiltertwithtnonspecifictDNAtortproteintandthybridizettotlabeledtprobe
-tdetecttbytautoradiography

, Explaintthetprocesstoftreplicationtelongationtandtalsothighlighttthetfunctiontoftprimase,tSSBt
andtwhatthappenstaftertantOkazakitfragmenttistsynthesized?t-tCORRECTttANSWER-
Continuoustcoretpolymerasetleadingtstrandtsynthesis
LaggingtstrandtOkazakitfragmenttDNAtsynthesistintprogress
PRIMASEtenterstastcoretpolymerasetnearstcompletiontoftOkazakitfragment
PRIMASEtmakestthetRNAtprimer
SingletstrandtbindingtproteintSSBtprotectstDNA
NewtβtclamptloadedtontotlaggingtstrandtattthetRNAtprimer
SynthesistoftantOkazakitfragmenttcompleted
Laggingtstrandtcoretpolymerasetreleasestthetβtclamp
Laggingtstrandtcoretpolymerasettransferredttotnewlytloadedtclamp
Oldtclamptabandoned
LaggingtstrandtcoretpolymerasetsynthesizestnexttOkazakitfragment
βtclamptloadertbindstandtopenstnexttclamp

Explaintthetstepstoftsitetdirectedtmutagenesis.t-tCORRECTttANSWER-Steps:
ssDNAtfromtBacteriophage
Heatttot55tºCtandtcoolttotroomttempttotannealtmutagenictoligo
tOligonucleotidetmusttbetphosphorylatedtont5'tend.t(TreattwithtT4tPNK)

tKlenowt(DNAtpolymerasetI,tLargetFragment)thast3'--

>5'tproofreadingtexonuclease,tbuttnot5'-->3'texonuclease.
AddtKlenowtandtdNTPstheatttot37tºCtfort1-
4thours,twhichtallowstthetpolymerasettotproceedtaroundtthetssDNAtfromtBacteriophage
NotetthististjusttonetroundtoftPCR
LigatetendtwithtT4tDNAtLigase,tbecausetittwilltstop.tCalledtheteroduplex,tbecausetthettwotar
ettogether,tbuttdifferent
ThenttransformtintotE.tcolitcells
Thentyoutgettsegregationtoftmutagenictandtnon-
mutagenicthomoduplexestoccurstaftertreplicationtintcells.

FunctiontoftDNAtPolymerasetGammat-tCORRECTttANSWER-
ReplicationtoftmitochondrialtDNA

Givet2tdisadvantagesteachtfor
t-tBACt-tCORRECTttANSWER-

1)tmoretdifficultttotmaketlibrarytthantwithteithertlambdatortcosmidstdifficultttotmaketandtmaint
aintlibraryt2)trecombinationtwithintthetclonedtDNAtistatpotentialtproblemt3)tDifficultttotpurifytsi
gnificanttquantitiestoftclonetDNAtduettotlowtcopytnumber

Givet2tdisadvantagesteachtfor
t-tYACt-tCORRECTttANSWER-

1)tdifficultttotmaketandtmaintaintlibraryt2)tchimerictinsertstaretatsignificanttproblemt3)trecom
binationtistatsignificanttproblemt4)tVerytdifficultttotisolatetandtmanipulatetclonedtDNA.tDifficu
ltttotseparatetclonedtgenomictDNAtfromtYACtvector.