Constructing and Screening Normalized cDNA Libraries - Google Sites

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Constructing andScreening Normalized cDNALibraries MARCELO BENTO SOARES ANDMARIADEFATIMA BONALDO A CDNAlibraryis consldered normalized whenthe frequency of eachCDNAin the libr¿ry is equaly represented reg¿rdless of whetherthe cDNAis derivedfroma rareor a frequently occurring ñRNAspecies. Theffequencies of all clones aretherefore withinthesameor, der of magnltude rángein a typic¿lnormalized library(see,e-9.,Soares et al. 1994; Bonaldoet al. 1996).This chapterprovides detailedprotocols for construction and normaizatlonof oligo(dTfprimed, directionally clonedcDNAlibraries in ph¿gemid vec (Okay¿ma tors. l\4odiflcations in the existingmethodsfor libraryconstruction and Berg 1982;Gublerand Hoffman1983;D'Alessio et al. 1987)andsolutions for the problems thataremostcomr¡only observed in cDNAlibraries arediscussed. Theseproblems are(1) the highfrequency of cloneswithsmallinserts, someof whichcontainnothingexcept the poly(A) (2) the presence tailsof mRNAs, of a longpoly(A) tail at the 3' end of cDNAs, whichnrakesit difficultto obtain3'terminal sequences, and (3) the occurrence of chir¡ericCDNAclones.In addition,a protocol for screenlng cDNAlibrariesby coony hybridization is presented.

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Primaty UsesandApplications forcDNALibraries

a cDNA librarl requireconrrucling and screenhrg bur ins!cadmay simplyinvolvesearchinglhe pubhonologous!o !he onc lic databases ior sequ€nces

AND EXPRESSION STUDYOF GENESTRUCTURE One of rhe {irst stepstoward cloning a gene oi inlerest typically involves construc¡ing a CDNA library f¡om a tissue or cell type likely to expressit and screening lhe library eirher by hybridizarion (Young and Davis l983a,b; Huynh et al- 1985, Sa¡¡brooket al. 1989)or by PCR (FrohmaneI al. 1988;Saiki et al. 1988;complon 1990;Frohman 1990). AfIer a cDNA clone is isolatedand se quenced lor verificalion tha! it correspondsto the desiredgene, it is ¡hen used as a probe ¡o screena genomic library and lhc¡eby to isolate ils cogn¿¡e gene.It is alsousedfor northernan¡lysisio study r L rp d ¡ l ( r no f e r f r e . ' o n n d r i t e . ( n cr e | . y p e . .r i ' sues,and stagesof developmen¡. The comparativeanalysjs of lhe CDNA and ils gcnomic cione r€veals,al l€asl in corresponding p ¿ - r . r h e \ r r u L t u r eo L " e g e n . . H L ' w e v e l .i " e cDNA clones often representincomplele producls of ¡everse transcripiion, characterization of the enlire transcript frequently involves additional sc¡eeningsof CDNAlibraries to isolate overlapping cDNAs.Alte¡natively, the m¡sing CDNAlragments can be obrainedby RT-PCRapproaches 15' and 3' RACE) with specificoligonucleoddesdesignedon lhe basisol sequenceinformalion derived irom the truncat€dCDNA (Complon 1990;Frohman 1990; seealsoFanningand Gibbs1997).

OF GENESBY PARTIAL RAPIDIDENTIFICATION SEQUENCING OFcDNAs An alternative approach lor gene discovery has emergedin the pas¡yearsthat is expec¡edto lacilirate quite dramalically ihe rvork involved in cloning a gene lAdamr er at. 1991, 1992, l993a,b) Khan e¡ al. 1992; Okubo e¡ a]. 1992; Malsubara and okubo l99l; M¿roba eI al- 1994; Okubo 1994).ln this approach,large numbersoi di¡ec: tionally cloned cDNAs arc randomly chosen from librariesand sequenced i¡om onc or both (l' and ' 5 ) ends.Their nucleotide sequences,and rranslaiions thereof, are comparedwirh those availablein public da¡abasesior idcnriiication of homologies lhat occasionally eiLhe¡ reveal or suggest ¡heir fLrncaion.li is anlicipatedlhat once most genesare identified and lhei¡ cloneEbecome available, lhc p¡ocessoi isolaling a CDNA clone may no long€r

Overview of the Methodsfor Double-stranded Synthesizing cDNA hasbeen madesincerh€ firsl meI:r' Much progress cDNAs was repo¡led(Ehlraliadise: od for cloning "t.

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proiocols, the hairpin-loop struclure narur¿,. iorrneda¡ the l' end of lirsl-s!¡andCDNArs u-.:: ¡o selfprime lhe synthesisof a secoodstrandaj:.: hydroiysis oI thc RNA remplate.The end prodü.i:: DNA ivirh the 5' end :: a hajrpindouble'srranded ' ' , . I R \ A e q J e ' r , ei n r h , f o ' n o f J j n . i . .r'oeo loop ot vdfldblr n/e dnd loc"..on T' rcsul¡ing cDNAs are then trealcd wilh nucleasej r o d r E € .rrh ( ' r n g e - . r . r r r d eDd N A l u . l J r e p : ' : . invariably removes portions ol thc cDNA i:.' r e . p o n d i n rE. , o J r n g o r . ' - r . o n . o d r n ;r . ; ' ¡ rhe mRNA. TJ {ir,,rrvcnr I| F need lor J,; 'r,on . ' n u c l e d \ es l . | " n d e r J l \ l o 8 | ) d e \F l o f ' i€ d l t F r n ¿ te: \p r o L oJ,l I n { h r . h ¿ h o r n o p o l v ' n e : . . is addedto the l' end of !]refirf's¡rand cD\_A=:: ' ' r ' e dl o : : . ' o n p l e | |e n l . r ! u l r t ñ n u . e o r i d e second-strand syn¡hesis. I n r l _ e icr ¿ s 5 rp. " p e r .O k d \ d m rr ' l d B e r ' : : : , l ( \ . f i 1 ( dr . l e l e g r n r- n F . h o dt h d r I n i c d " . , r CDNAbr -,li - . idea of synrhesisof second'strand primed nick translation.In this method, ,l : . I n R NA i r e v . r ' e | | d n \ ( , i b e d i I . o , D N A ' ) . r d i l ( dc l L ' n r nvSc c r ñ r 5 p r j ¡ ¡ e - .' 2 ' " l r n l ' . n r n r r \ l r g d L r l,oi r h ( D \ A : R N A l \ b r r d J : . . : . to ensüretheir circularizadon, and (l) lh. ::::: : s , r , n d i \ . t n r h e . i / e d b v r . : n L ir c o r ' b ' : : RN¿seIl, ¿. roli DNA polymerasc I, ¡nd E ji.. -l . \!::. -ri. ligaseto replacelhe RNA iD lhe hyl.Jrids : R t \ r \ e H r \ ¡ n e n d u r i l , o n u . l eF". p e . i t : . : . : . R N A . r r ¿ . l do f d D N A R N A h y l , ' J l 0 7 t l . T h i \ e n d o r ; b o n J ' l e " rI n ( f i u d u .), - . - . . : smallgapson ¡he RNA frand, rvhichafe.'::::::: { e r u n L c l ( , i lr}: . r . r ' \ I \ by rhe s O N A I o l y m e r d \ eI ( n r c k U - n 1 , , : o D N As r r d n d i , \ y n h e \ i / e d b \r h e o u l , n . ( : . , , Guble¡and Hoiiman (1981) describ.: = , :i: -:: m o d i l i c ¿ . ' o on i t h r O k d ) " r r " r ' r B r

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prorocol that combjncsthe classicoligo(dT)-primed íir5t s!¡and synthesiswilh the novel RNaseH-DNA poiymerasei-mediated second'strandsynihesis. Homopolymericrails a¡e added to the ends of both rhe cloning vector (dCTP tail) and the cDNAs,. (dcTP tail) and then ligated.The advantageof this procedure is lbat neither the elaborate Okayama and Berg vector-p¡imer system nor lhe classic by nucleaseSl js used. htri¡pin'loopcleavage A limitation of úe RNaseIl DNA polym€raseimediated method ior second-strandsynthesis is ¡bat ii results in the loss ol sequ€nceinlo¡mation from the 5' end oi thc mRNA template (D'Al€ssio ¿nd Gerard 1988).This can be explainedas follows- Removal of the 5' mor RNA primer by the combination of t. ¿dli RNase H activity and lbe '-Jl' €xonuclcoiyticactivity of -6.col¡DNA poly5 m€raseI mak€s the complcmentary dNTPs wh;ch are located at the 3' end oi a full'length ii$tstrand CDNA, single'sirandcd. Single-stranded DNA, in turn, is hydrolyzed by the I -+5' exonucleolytic activity of E. coli DNA polymeraseI, leaving the end ot the doublc-strandedCDNAproduct blunl or nea¡ly so. Therefore, du¡ing subsequent cloning, sequence information correspoDdingto the 5' most RNA oliSonucleotide that primed se€ond-strandsynrhcsisis ]ost. However, this loss c¿n be minimized by adding RNaseH alter, ;nstead oI wit]n, E. coli DNA polymeraseI in the second srrand synth€sis ¡eaction (D'Alessio and Gerard 1988) sin€e RNase H is not capable ol cleaving hybrid RNA at thc junction belween a ribonrrcleotide and a deoxydbonucleotide (Omer and Faras 1982;Resnicket al. 1984). For furiher discussion and figures illustrating these methods lor synlhesizjng double-stranded cDNA,seeSambrookct at. (1989,Chapter8)

CloningVectorsfor Construction of cDNALibraries Ther€ are two main choices of cloning veclor for construciion ol CDNA liblaries: bacteiophage ¡' v€clors (Murray er al. 1977; Young and Davis 19sla,b;Huynh et al. 1985;Shortet al. 1988tPalazzolo et al. 1990) and plasmid vectors (vieira and Messing1982,1987,t99l; Mead et al l98ó; Dentc and Cortese 1987; Heidecker and Messing 1987; Mead and Kempe¡ 1988). Both cloning systems have advantagesand disadvantáges,and the best

choice !vi1l greatly depend on the specilicapplica'

A VECTOR IN CHOOSING PRINCIPLES GENERAL Cloning cfliciencies (as deiined by the number ol plu or cfu per microgram of bacteriophage;\. or plasmid.vectorDNA, respectively)are no longer a factor in deciding whicb cloning vector to use With the advenl of electroporaiion (Dower et aL. 1988), cloning ellicienciesof recombinant plasmid DNA became comparableto those atiained bY in vlro packaging ol recombinant bacte¡iophageI DNA. Efficiencies of in vitro packaging (using cigapackII Gotd Istratagene2002141)and electroporation (using Electromax E. rolt DHIoB [Life are very similarfor con18290-0151) Technologjes rrol DNA (2 x l0e pfu/lrg ot wild'rype bacreriophagel. DNA and I x l0r0 €Iu/pg of CsClpurified supercoiledpUCl9 plasmid DNA) Thus, a typical ligation containing 50 ng of CDNA (with an ave¡agesizeol I kb) and a twofold mola¡ excessol vector DNA (5 ftg ol bacteriophage¡' DNA or 0.1 pg ol puclg DNA) would be expeciedlo yield approximately three limes more recombinantswith the bacteriophagevector than with the plasmid Bacteriophagel libmries are easier ro screen than plasmid librañes. Mo¡e clones can be screened per plate, replica filters can he more rapidly prepared, and the hybridizarion back ground tends to be lower. Hor4ever,plasmid veclors offer greater versatility than bacteriophage¡" vectorsfor subseqüentmanipulatlon. For examplc, a plasmid DNA library can easily be purified by agarcsegel electrcphoresisto eliminate all of the nonrecombinant vector molecules as lvell as any clones with small inserts that óay have escaped size selection isee pp. 121-l2l). This type oi purilication cannot be as easily accomplishedin bacteriophagc1" iibmries. From a technical sland' point, it is therefore iar easier to obtain 8¡eater - yields of individuaily selectedplasmid DNA than bacteriophagel DNA for subsequentsubcloning and manipulation of the insert I VECTORS BACTERIOPHAGE The most widely used bacteriophage)" vecto¡s for CDNAcloning are ¡,gilo, ¡'8tI l, and lambda ZAP. These bacteriophage), inseÍion ve.lors tl?ically accept inserts of 0-10 kb. Lambda ZA? (Strata-

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GDNALibraries Normatized andScreenins 2 r construct¡ns ChaDter

gene) has some advantagesover the other bac¡eriophdgetr cloningvei LoI.bec¿u.eir contd;n'J pld'mid component (pBluescrip!, which has an fl origin oI replicalion and a polylinker wilh multiPle cloning sites) that €an be excisedin vivo after s perir ec¡ion wiü a helper bacteriophagean'l lhen recirculariued,thus eliminating the time involved r'e-s¡er Ld in subcloninS . m b d ¿Z A P i s l h e r c f o r e vecror with which to work. VECTORS PHAGEMID The obse¡vaiionby Dotto er al (1981) thar a plasmid carrying the intergenic region of the Iilamenlous bacteriophageIl can be packaged as single'srrandedDNA into a viral parlicle by a her per bacteriophageled to the construcrionof ve'tors (phagemids)üat combine rhe advantagesof both plasmid and bacreriophagesysiems (zincler and Boeke 1982; Mead ánd Ke¡nper 1986). The p¡ob lem originauy encountered in the use ol these phagemids was the significant reducrion rn tlle amounl ol single-franded DNA próduced as compared io bacreriophage vectors snch ás bacMll. teriophage e) r h ( T h 1 5r e d u ( r i o nw d s d u e r o i n e r f F r e n , b phagemid wiih the replication ol the helper bacteiophage (Enea and Zinder 1982) This problem has now been solved. Wi¡h lhe use of bacteriophage mutants such as bacleiophage MrlKOT, which p¡eferenlially packagesplasmid DNA over bacteriophageDNA (Vieira and Messing 1987), suilicient amounts ol single-strandedplasmid can be ¡outinely obiained irom small-volume cultures il pg ol single-sr¡andedplasmid DNA per millilirer ol culture). A delailed prorocol for con' srruction of tDNA libraies in phagemid vectors is providedon pp. 8ó-123.

for of the Procedure Overview DirectionallY Constructing ClonedcDNALibraries An outline of ihe stepsinvolved in the construc_ tion of direcrionally cloned CDNA libraries in phagemid veclors is depicredin Figure I A r¡o¡e derdrleddenriplion ol rhe processof cloni¡g d bona fide CDNAis presenÍedin Figure 2 and in the protocol on pp. 8ó l2l. Bdeily, a Nr{-(dT)ls oligonucleotide(i-e, an

oligo(dT)t8 flanked by a Nr¡I restrjction srte at lts 5'end) is usedto prime rhe synlhesisoi firsast¡and CDNA wirh RNase H- reverse tra¡rscriptasefuom MMLV (Liie Technologies).(This enzyme is ¡ecommended for ils ability to generate longe¡ reverse lranscriplslhan mos¡ commerciallyavailablelorms of reverse transcriptase,) "one-¡ube" first- and second-strandCDNA synlhesesare perfo¡med essenrially as describedby D'Alessio et a1 (t987) Double-srrandedcDNAs thus genera¡edare lreaied with bacte¡iophageT4 DNA polymeraseto c¡eate blunt ends, size selecredby gel filtration on a Biocel A-50ñ (100-200mesh;Bio'Rad)column,and ljgatedto a largeexcessof adaplermolecules(e g, EcoRl adapters). Adapterligaled cDNAs are then digestedwith No¡I and purified on a sepharoseCL 48 (Pharmacia) column to remove the excess adapter molecules.Size-selecredcDNAs (>350 bp) are úeated wilh bacteriophageT4 polvnucleotide kinase Io phosphorylale the adapler ends (one ol ¡he two oligonucleotidesol the adapter molecule hd. a s'-hydroxyl groJp ro Dfcvcnl concdlemeri_ zation oI adaplersduring liSalion) and ligated dire.Iionally to the NolI- and E oRi-digestedends of a phagemid veclor (pT7Tl-Paq for veclor des' € ption, see p. 86). Bacreriaare rransformedwilh rhe pudlied ligatedma¡erial by electroporalionand F r o p ¿ g " r eudn d e r . e l e , r i o nw : r h d n " p t r o p r i . r e antibiotic.Finally, plasmid DNA is prepared. A recommended option ar üis pornr rs 10 eliminale from lhe libmry all ol the nonrecombinani ("enrp¡y veclor") molecules (seepp. I l l . l 2 l ) ¿ < f o l ' o w \ :A p l " ' m r d D N A p r e F d r ¿ r ¡ o n oi the library can be linearized wiih N¿¡I and the recombinantDNA separatedlrom the emply vecror by agarose gel electrophoresis The recombinanl DNA is then purified using B-agarase(New England Biolabs) and recircülarizedin a large-volume ligadon. The recircülarized DNA is precipiared. bacteria are úansformed by eleclfoporation and L n o e r s e l c c r ' o nw i t h ¿ r ¿ p p l u p r i ¿ l e frop¿gdred antibiotic,and plasmid DNA is prepared

Commonly Problems in cDNALibraries Encountered La4e-scale sequencing of cDNAs has uncovered some of the probtems commonly encountered ir oligo(dT)-primed,directio¡ally cloned CDNAlib|arj e ) ( A d ¿ m \e l a l . l q e l . l o 9 ' l L \ o d r ( 5 l 9 q 4 l ,5 u ( ü as rhe high frequencyof cloneswi¡h a long polytA,

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€ rm e r l o ¡ { d T ) t so l i g o n ueco t ¡ d P S y ¡ t h e s ¡ zf ie¡ s ls t r a n dc D N Au s l ¡ g ¡ranscrprase reverse l s y n t h e s i zsee c o n sdt r a n dc D N Ab v n l c k e a n dE c o l ¡ I t r a n sa t o n u s i n gR N a s N D N Ap o l y m e r aIs e t i t hb a c t e r i o P h aTg4eD N A I T r e aw I Poiymer¿se B l u n te n d e dd o u b l es t r a n d ecdD N A l s z es e l e cbt yc o u m nc h r c m a t o g n p l r v S z es ee c t e dd o u be s r a n d € dc D N A( > 35 0 b p ) L gateto excessEcrRladapters Ligaiedc0NA/adapier I D s e s tw t h l o ¡ l i o a l l o wd ¡ r e cot ¡ a l c l o n r n s

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an'l a'rapter excesslree ¿dapters I Remove c h r c m a tosmPnY b Y c o ! m ¡ d i m e r s { aód¿oo nC IPocpor¿." ho eoo fe4 p o l v n u c l edoei k ¡ a s c b a c t e o r p h a s i 5' e¡d wiih plrosphorvlated Ligate¡cDNA/adapter r N Aa n dc D N A / a d a p t e r L ¡ C a tvee c t oD I l NA c D N A / a d a P tgear t e dt o v e c t oD ( u n a m Pi e d c 0 N Al ¡ b r a r y ) and I lransform6 col/ by e ecfoporatLo¡ qe biotic + p r o p a c aut en d e r e c t i o nw t h ¿ n t P r e p a rPel a s m i0dN A P l a s ñ i dD N AP r e P a roáni ] oigestwith ruot I

. u é P I i 1 u c P ¿ g " r d" r or " - o " m r . o - b r o n rv o ' - o m oo u r ó \ { mt o l e c e us z er e c o ñ b i n a n lReci¡cula, R e cr c u l ¿ ¡ i z eP du , r i l i eP d a s md D N A on and fransiorm E coli bv electrÓporai I p r o p ¿ c a t e s e l é c t l o w n ¡ | r a n tb i o t l c u ' d e r + P r e p a fPei a s m i D d NA t P ¡ a s m ¡DdN A( a ñ p l ¡ f i ecdD N A b r a r vr e a d v _ tnoor r ñ a z a t i o n ) CDNA libraries in Figlre 1 Flow chart of the steps involved in the construction of directionally cloned phagemid veclors. See protocol on pp 8ó-l2l for dctails rail and ih€ occurrence of chimeric clones (ie., CDNA Iragmenls derived frcm differenr mRNA molecules that are join€d by ligation in a single CDNA clone). Ahhough the lormer poscs limitaoi r.q.,..t." Irom ihe l' end i¡"t f"i g."*"1." of a cDN¡, the lalt€r is evcn more serious since it oitcn cannot be idenriljed and may rh€refore resrrlt

ln addition' CDNA in mislcading inlormation clones with small inserts' sometimes conslstrng exclusively oi pieces of mRNArails' prevail in manv l i b r a r i e s( A d a m s e t a l l 9 9 l ' 1 9 9 2 ) T o g c ¡ h e r 't h e s e undesirablc features represent maior impediments to the process of obtaininS thc compl€le nucleotide sequencc ol a transcrrpl'

cDNALibraries Nolmalized andScreening Chapter2 ¡ Construct¡ng

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