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Lesson1DNA Templ at ePr epar at i onFocusQuest i ons 1. Whyi si tnecessar yt oc hel at et hemet al i onsf r om sol ut i ondur i ngt heboi l i ng/ l ys i sst epat 100° C?Whatwoul dhappeni fy oudi dnotuseachel at i ngagents uchast heI nst aGene mat r i x ? I ti sne c e s s a r yt oc he l a t et heme t a li o nsf r oms ol ut i ondur i n gt h i ss t e pbe c a us et heme t a li onMgi sa c of a c t orf ore nz yme s . I fMgi spr e s e nt ,e nz yma t i cr e a c t i onswi l la c t i v a t et hee nz ymeDNa s awhi c hwi l l de gr a det heDNAa st hec e l li sl y s e d.I ft hei onsa r ec he l a t e d,t he ywi l lno tbep r e s e ntdur i n gl y s i st oa c t i v a t e DNa s ea ndnoneoft heg e no mi cDNAwi l lbede gr a d e .We r et hec he l a t i n ga g e ntno tpr e s e nti ns ol ut i on, DNa s e swoul dbea c t i v a t e dduet oMgs t i l li ns ol ut i on,a ndt heg e nomi cDNAwi l lbede gr a de d,a ndt he PCRwi l lnotoc c ur . 2.Whati sneededf r om t hecel l sf orPCR? Theg e nomi cDNAi sr e q ui r e df ort hepo l yme r a s ec ha i nr e a c t i ont ooc c urs i nc ePCRa mpl i fie st heDNA s e que nc e s .I ft he r ei snoDNAt oa mpl i f y ,t he r ei snoPCRr e a c t i on. 3.Whatst r uct ur esmustbebr ok ent or el easet heDNAf r om acel l ? Thes t r u c t ur e st ha tmus tbebr oke nt or e l e a s eDNAf r omt hec e l l sa r et hec e l lme mbr a nea ndt he nt he nuc l e a rme mbr a ne . 4.Whydoy out hi nkt heDNAi sst or edcol dwi t ht heI nst aGenemat r i xaf t erboi l i ngt hesampl es ? DNAi sst or edi ni ceaf t erboi l i ngt osl owdownandDNasest hatst i l lmayr emai ni nt hesol ut i on becausenotal l t hemagnesi um wasr emov ed,sot her est i l lmaybeact i v eDNasest hatcoul d degr adet heDNA.

Lesson2PCRAmpl i ficat i onFocusQuest i ons 1. Whyi si tnecessar yt ohav eapr i meroneachs i deoft heDNAsegmentt obeampl i fied? Thet wopr i mer sal l owf oranexactsequencet ober epl i cat ed.Theyl ookf oranexactsi gnalona DNAsequence,andoncet heyfindi t ,t heybi nd,r ecr ui t i ngDNApol ymer aseandbegi nr epl i cat i ng. Theyar et hest ar t i ngpoi nt sf ort hi sent i r epr ocess. 2. Howdi dTaqDNApol ymer aseacqui r ei t sname ? Taqpol ymer as eget si tnamef r om t heheat s t abl ebact er i um i twasf oundi n,Ther musaquat i cus. I tl i vesi nhi ght emper at ur es,whi c hal l owsi tt obeagoodagentf ors ynt hesi zi ngDNAdur i nga PCR. 3. Whyar et her enucl eot i des( A,T,G,andC)i nt hemast ermi x ?Whatar et heot her component soft hemast ermi x,andwhatar et hei rf unct i ons ?

Nucl eot i desar er equi r edi nt hemast ermi xbecauset heyar eoneoft hesubst r at esf orr epl i cat i on t ooccur .Wi t houtnucl eot i des,t her ewoul dbenot hi ngf orpol ymer aset oaddt ot hegr owi ngchai n andr epl i cat i onwoul dnotev enbegi nt ohappen.Theot hert hi ngsi nt hi smi xar eTaqpol ymer ase, whi chaddst henucl eot i dest ot hegr owi ngc hai n;magnesi um i ons,acat al y stusedbyDNA pol ymer asei nor dert oel ongat et hest r ands;ol i gonucl eot i depr i mer s,compl ement ar ysequences ofDNAt hatwi l lannealt oanex actpoi ntoft het empl at ewher ei ti sneededt obegi nmaki ng copi esofanex acts equence;andt hebuffer ,whi chhel psmai nt ai nt heappr opr i at eenvi r onment f ort hePCRt oocc ur . 4. Descr i bet het hr eemai nst epsofeachcy cl eofPCRampl i ficat i onandwhatr eact i onsoccurat eacht emper at ur e. Thet hr eemai nst epsar edenat ur at i on,anneal i ng,andext ensi on.Denat ur at i oni nvol v est he separ at i onoft hedoubl est r andedDNAt omakesi ngl est r ands .Themi xt ur ei sheat edahi ght emp f ort hi st ooccurf orashor tt i me.Thi sst epi si mpor t antbecauset hepol ymer asecoul dnotcomei n andannealt ot hepr i mer sandmakesnewcopi esoft heDNAsequences.Thebestt emper at ur e f ort hi spr ocessi snor mal l yar ound94degr eesCel si us.I nt henextst ep,anneal i ng,t hepr i mer s anneal t ot hei rcompl ement ar ysequencesont heDNAt empl at esot hatt hepol ymer asecanbi nd t ot hem andst ar ts ynt hesi zi ngDNA.Thebestt emper at ur ef ort hi spr ocessi susual l yar ound60 degr eesCel si us.Thel astst epi st heext ensi onst epwher eTaqpol ymer aseaddst henucl eot i des t ot hegr owi ngst r and.Pr operaddi t i onoft hesenucl eot i desi sar ound74degr eesCel si usbecause t hati swher et hepol ymer aseact i vi t yi sati t sbest . 5. Ex pl ai nwhyt hepr eci sel engt ht ar getDNAsequencedoesn’ tgetampl i fiedunt i l t het hi r d c ycl e.Youmayneedt ouseaddi t i onalpaperandadr awi ngt oex pl ai ny ouranswer . I tdoesn’ thappenunt i lt het hi r dcy cl ebecauset her ei snosuc ht hi ngasa“ st op”pr i mer .Fort he fir stt wocy cl esofPCR,t hepr i mer sar eext endedcompl et el yunt i lt hepol ymer as ef al l soff becauset her ei snot hi ngt os t opt hem f r om goi ngonandon.Thepr i merwi l l annealt ot he sequenceandext endsf r om 5’ t o3’ ,l eavi nga3’ ov erhangbyt het i met hatpol ymer asehasf al l en off.Thesamegoesf ort heot herst r and.Thenextpr i merwi l lanneali nt hesecondst ept ot hat r epl i cat eds equenceanduset hatast henewt empl at e.I tt henwi l lr ecogni zet he3’ OHont he t empl at e,anneal ,t hepol ymer asewi l lcomei nandt henel ongat eagai nf r om 5’ t o3’ ,t hi st i me, l eavi ngt he3’ov er handunr epl i cat edandst oppi ngatwher et hel astpol ymer asest opped.Nowi n t het hi r dcy cl e,t hepr i meragai nfindst he3’ OH,pol ymer asei sr ecr ui t ed,andal lt her ei sl ef tt o r epl i cat ei st heexactsequence.Thi spr eci seampl i ficat i onoft hesequencecanonl ybedoneby t hesespeci ficpr i mer s.

Lesson3GelEl ect r ophor esi sofAmpl i fiedPCRSampl es FocusQuest i ons 1. Ex pl ai nt hedi ffer encebet weenani nt r onandanexon. Ani nt r oni sanonc odi n gs e que nc et h a twi l lr e ma i ni nt henuc l e usbe c a us ei ti ss p l i c e da wa yf r omt hee x ons . Ane x oni sac odi n gs e que nc eofDNAt ha twi l ll e a v et henu c l e usa f t e ri ti ss pl i c e da ndwi l lul t i ma t e l y be c omet heme s s e n g e rs e q ue n c et ha tc ode sf orap r ot e i n.I nt r onswi l ls o me t i me sv e r yi nt he i rs i z ea nd s e que nc e ,bute x onswi l lno t . Thee x on sonl yc ompr omi s ea b out5% oft heDNAwhi l et hei nt r onsma keup t heo t he r95%. 2. Whydot het wopossi bl ePCRpr oduct sdi fferi ns i zeby300basepai r s ?

Theydi fferby300basepai r sbecaus et hebandwi t h941basepai r scont ai nst heAl ui ns er t becausei tt hebandt hati ndi cat esAl ui spr esenti nt heDNAsequence.Theot herband,641,does nothav et heAl ui nser tt hati s300basepai r s,sot hati swhyi ti sl acki ng300. 3. Ex pl ai nhowagar oseel ect r ophor esi ssepar at esDNAf r agment s.Whydoesasmal l erDNA f r agmentmov ef ast ert hanal ar gerone ? Theagar oseel ect r ophor esi ssepar at esDNAf r agment sbasedont hei rsi zes.DNAi sl oadedi nt o t hewel l s,andast heel ect r ophor esi si sr unni ng,t hedi ffer entDNAf r agment sbegi nt o“ r un”down t hel ane.Thebi ggerf r agment sar eheavi erandcannott r av elasf ar ,butt hesmal l erf r agment sar e abl et ot r av eldownt hel anef ar t her ,t hussepar at i ngt heDNAf r agment sbyt hei rsi z e.Thesmal l f r agment smovedownf ar t herbecauset heyar eabl et omaneuvert hr ought hegel muchbet t er t hant hel ar gerones,sot hel ar geroneswi l lj ustst opwhi l et hesmal l erf r agment sk eepgoi ng. Theyar eabl et omovedownt hegelbecauset heyar edr awnt owar dst heposi t i v epol eoft hegel anddr i v enawayf r om t henegat i vepol ebecauseDNAf r agment shaveanegat i vec har ge. 4. Whatki ndofcont r ol sar er uni nt hi sexper i ment ?Whyar et heyi mpor t ant ?Coul dot her sbe used? Ther ewer efiv econt r ol st ot alt hatwasusi ngdur i ngt hi sexper i ment .Thefir stt hr eecont r ol swer e t hehomozy gouscont r ol s[ ( +/ +)and( / ) ] ,andt hehet er ozy gouscont r ol( +/ ) .Thesecont r ol swer e usedt obeabl et or ecogni z ewhatahomozy gousbandwi l ll ookl i kei ft heAl usequencei spr esent i nbot hchr omosomesornot .Het er ozy gouscont r olwasusedt oshowwhatt hel anewi l ll ookl i k ei f someonebot hcodesf ort heAl ugeneanddoesn’ tcodef ori taswel li nonechr omosome.The ot hert wocont r ol swer et he“ not empl at e”cont r ol s.Thesewer eusedt oshowi ft her ewas cont ami nat i oni nt her esul t sbecausePCRi ssosensi t i ve,andcont ami nat i oni seasyt ocont r act dur i ngt hesel abs.I ft her ewasabandi nt hesel anesot hert hant hepr i merdi mer ,whi chi snor mal , i ti ndi cat est hatt her ewascont ami nat i on,andt her esul t smaycont ai ner r or s .Theset wocont r ol s hel pwi t ht hei nt egr i t yoft heexper i ment .Thet hr eecont r ol sofwhatcoul dbeseendependi ngon whi chc hr omosomest hest udenthav ewer enotr eal l ynecessar y ,butt heywer eagoodr ef er ence. The“ not empl at e”cont r ol scoul dnotr eal l yber epl acedbecauset heywer esoi mpor t anti n det ect i ngi fanyt hi nghadgonewr ongdur i ngt heexper i ment ....