BacterialCloningSystems KENDEWAR, IANDUNHAM, KIM,ANDMARKT. ROSS UNG-JIN of genomc n the developr¡eni Thefieldof ge¡omeanaysishasrecentlyseena resurgence jability oi re a¡d coning capaciv, fhe ease of application, DNAI brare; uslngE collhosts. jdea ysis ncludirg genorne experlmenls, ana most foT baclerialcloningststemsmakethem as comr¡on Theuseof theselibl¿rjes andgenor¡icsequencing. mapping largescae physjcal gridded seres of high ibraries and a of developr¡ent by the reóurceshas beenfacilitateci This chapter clones numbe|s of arge analyzlng protocos for hancllng ancl throughput repllcation, storage, procedures o¡, in the consiruct and thegeneralconsderaLions clescribes arebasedprimar' esd scussed andexamp librares.Thetechnlques a¡d anaysisoi bacterial genor¡e analys s' for lbr¿ries c ones and P1,BAC,andPAC fosr¡icJ, ilyontheuseof cosmid, syslem, cosm d of the manlpulation and clone on libraryconskuction Fordetaled Drotocols (lh s volume); ¿nd seeSternberg seeEvanset al. (thisvoume);for bacterophageP1vectors, (thls volurne). seeBiren et al. for BACS,

Clor¡ngSvstems Ch¡pterI ¡ Bacterial can be recovercdrepca¡edlyover a perjod ol year! l r o ' r ' b r . ' J r 'l l r r o J j h or l l l r er ( ' c " r ' 1 " ^ n n r r r i ty. In addj!ion, ltre addressesof arraved clonesca': cisilv be sharcd among ürvestigalors who háv' During 1he I980s, cloning vuct.rrssuch asplasmids' occeisto tbe safle library along with ilrfonnalior: bac¡criophagel, or cosmidswere used lor ihc cont11e about the sequenccslhey contaiu l¡ this way cla¡¿ strlrclion ol genonic DNA libraries Wilh abc'ütspecificcloncscar acclrmularea¡rdthe clonÉ\ exceplion ol systematicápproaches!o ihe analysis becorneeven more valuableihrough the 'ontrLblr of sonlesmallgenomes(coulsonet al- 1986;olson 'n J \v"\ : u n ^ l s n - \ r r \ i n , l I F r .n l I h u d r o r r c e! al. 198ó; I(ohara et al. 1987),mosl irvestigators ' n . ! ñ . . i t , , er a . r \ r ' u r r " r r d ) (g, 1i n o r ' ' . l r ¡ f i ' aimed at isolating a genomic lragmenl conla rng gerome analvsishai ihe adventof large-scale the gene or gene clr.rslerol intercst.Tbe small inserl been accompanicdby lwo major changcs]n ln¿ size-ofthesetypes ol clonesand the linliled siTeaDd way bacreial clonesalrd libr¡ries are uscd' Finr r1 mber ol the regions undcr invcsligationfavorecl tech¡ical advanccshavc produced a new g€nera the isolation ol cloncsby screeni g replicasol ran_ 'rg lhal are capableol clonirrggen(tur' Lionol v€cLors d o m l ) p l d r c ol i b r J e \ ' ( n ' r r L' r r r ' ba ' r ds e e t regio$ ol up lo 100 kb Thes€large'ürsen'lonlne theseIibrarieswas r1otdilficull, being accomplished veciors, including Pl (S!errberg 1990), BAL ¿ sl r e " d )i n r r ' - i r n - o ' rn ' o l ( ( u r l r u u e l rp r o c e d u r e (Shizuyaet al. r992), and PAC (loannou er ¿l l " i ! i ; 1 . ' ; y l ¿ b o , ¿ , . | | c rI n ¿ d d i r ir' ' l r c ' u ' r r r s t o q 4 , .d l t o sr J p . d d n . r ' y ' i . .r 1F g r ú r ' . ' p d n rgr -i r\ efficiencyfor thesesYslemswas high, thus, a single This providesLhemcans1o slud\ eral megabases. jnvesiigatorcorid rapidly creatcnew librarieswith (e.g., n1aln alian) gcnomes¡¡1'leLá! large even small amoünis ol DNA Hence, with thc focus otr second,thc needlo analyzc cLoDes. baclerial using ¡olating a specificregion ol iDter(st, thcre was lrt has spurredtbe deYcL syslemallcally regions large tle need !o maintain rhe libraries as permancnt ^ l l c r r n pJ ( ' l ; ' r ' r r r i ^ l g r ' d d . . l ' l ' f " r ' c ot mrnr n nlDerc)1org¡¡isnls lor ¿ (i.e., >l0x) coverage The abiljry ro clore megabaseDNA lrag¡nents of bacrerialclonin! the use in The resurgence asYACS(Iluke et a]. 1987) fundamenlallychangecl ol rlensc development due to the svstemshasbeen genome mappi¡g and analysis (see Green et al , YAC (botb fro physical maps ly popuLateclSTS lhis volume). But as valuableas YACSproved to lrc, mappürg ICollins cr ai basedand radiation-hybrid YAC library co¡struction was dilfi.ult aDd labori_ l997l) anJ et al 1995i Schulcr 1995;Hulisonel ¿1. ous. The p¡oduclion ol YAC libraries lirst involvcd jrrserr DN1 large for cloning the increasedcapacity masteriDga whole seriesol new techniques Alter of Plt capacity fraglllenls.Allhough the.loning these techniqueswere learn€d,a prolongedperiod 'oi rhal ol !o coÍrpared PAas. and BACSis lafge of "pro¿luction"work ensued,in which a re¡m ol H()wever YACs ihat ol rnids, it does rot approach investiga¡orswould perforrn ¡he same proceLlu¡es 1o tbe use ol bacteri¿ of thcre are severaladvamrages numbers sullicient reDeatedly Io accumulaLe clonesin los BacteiaL (loning sys¡emsover YACs. cl;nes to seneraie a librarl'. Th s, cach clore iu a g, PAC, and t()s¡lrkr BAC, copy numbcrveclors,e YAC library represenleda precious resoÜrcethat 'lrmrefr!n reduced are highly sLableancl sbow col d be recrcaledin only a lew iaboraloriesa 'l 'ell cu!' bacleri¡l compared1o YACSln addilio¡' even rhel] would require a seriorisinveslmerl ot yeaf cultLlrtr than ¡uresare easierto DraDipulale time and labor. When a library is arayecl. ' e, bacterialclonesaDd libraricsgrow norc rapr¡li ¡ individt¡anslomants original when each oi lhe than YACS,are casierio grid and handle, and gro$ ually picked into a singlewell ol a multiwell plaie, to higher celLdcnsi¡y,which allows ior nrore cIIi it becomesmore easily repli.aled, stored,scrcereci, higl Mof imponantlf reasonably cienl scrcening. add shared.In this wa, clones and libraries üat clon(' ¡luartiliesol in!act,pureDNA lronr bacterial would have otherwisebeen preciousor rare can be of lhe can be ¡olaled wilhoui lhe copu.ificaiionol ¡ !.: those outside by readily sharedand utilized genomicDNA, r.rsingsirnpleplas¡lid ba\ed DN: laboratoryin which rhe libr¿ry was ScneráredThe preparaLions. abiii¡y to distribute copics of lhc librades,and lhe Many investigatorswill n€ed !o coDslructnet has thclrl, ¡o screen fil¡ers ard DNA pools needed libraricslor ¡hcir.loning and mapprn: genomic obtain to our abili¡Y been a major conlribütor !o a growing rlrmber of iibrariesatu 'n Howevct 'lon€s stLrdies. the llecause and study genomic cloncs the loml of lrozeD plales t'i¡ available th€y readily permanent addresses, anayed libraries have

GenomicLibraries

Construct¡onof B¿cterialGenomicL¡b€¡ies

arrayedclones,iihers for scrccningby lrybridiza| ,n ,r LrNApour. ¡u D, P-h¿ed ..rperrrg Tl p 'rr'r lr. , \ r '\. j)ñ " , Pn i n v I ¡ b ñ d "\ ril"l roriesfrorn spendinglargc ¡n]olrnls of lime and rcsources recreating librarieslhá1afe slreadysuited cLoning 1()th€ir needs.therebyalloivingsmall-scale and mapping elfoÍs to ¡rakc use of the mof advanccd(looing 1€chniques.Cenonlic libra.ics arc olferingde€pcovcr¿gclrom a varielyot sp€¡:'€s LhfoLrgh ccntr¿ll¡cililicsor cornp¡now acccssiblc nies. The world wi(lc w€b addresseslor som€ ol thes€librarysoLrr(ciare listedbelow. Calilonia lnfitutc of Tc.hnokigy http: / /!¡tu, Lreé..aLtech. edu C l c I n s o nU n i v e r s i l y , G e r o m e C r ü t ( - ' f h t i p : / / g e n o n e . c l e m s . r ¡ . . : ( l u /a d d e x . h t r ¡ 1 R o s w e l l P a r k C a n c e rI n s l i t L r t c h t t p : / / b i c p ¡ c - r n é d b. u I f a l o . e d u Genonc Syslflns Ir1c. h t t P : / / r . d - q e d o m e s Y s t e m s .c o n / R e 5 e a r c hC e r € t i c s http: / /lÑv, rc:jqcn. co¡r/ German Hrnran Gcnorne Project: I{ZPD h t L r r: / / ! / w ! . r z p d . d e /

of Bacterial Construction Libraries Genomic Import¡nt f¿clorsto bc consider€dwhcn colrstrücling or rvorkiDgwilh librariesfor genomewide below.For detailedprolo aoalysisarc summariz€d cols on libr¡ry confrr.rction using the cosnid sys rem, seeEv¿nsct ¿1.(rhisvolume); lor bacteriolhage Pi v€ctors,scc Srernberg(thisvolumc);and Ior BACS,seeBirren ct ¡1. lthis volunre). BACTERIAL ERISTICS OF DIFFERENT CHARACÍ CLONINGSYSTEIIIS The divcrsily ol bacteial cloüúg systemsavailable for use in vaúoLrsstráinsof ¡..oli enablcsinvcslig¿lorsto choosr thc chning systembcst suitcd lol stecilic e¡perinrcnls.Table I summarizcs¡ va¡iety ,' v,.ror. r.Fd ,r' Jrl r¡Ir ,r.ter,l lorirg.). rems and d(scribesthe leatur€sol these1'€ctors rommonly sedin genomeanalysis.

Until the developmentol thc ncwcr cloniitg syslcrns,cosnids were widcly uscd.for cloning largc-insertDN,^.hagmentsin ¡. c01i.Cosmi.lshave lhe adv¿¡l¡ge of the high efficienq, oi ¿rr-¡¡rcdi¡, packaging.ht addition,cosa¡ed baclcriophage ol a sm¡ll vcctor(c.g.,a n¡idshavc the advart¿:jes pl.snlid) bccauseol recir{ulariz¡tion c¡ilhc DNA to a plasmid forr¡ Írirhin lhe host cell. Cosmidsallow rhe cloningof l5 -45 kb ol genomicDN and have beru üsed to build physic¿lr¡r¿psco¡rposcdol overlappirg cloncsin hLrm¡n genomesand those o{ orher organisms.Cosnlidsexist as multiple copies per cell, wtrich ¡llows recov€ryo{ large amounrsof DNA during DN,{ pr€paralio¡ lrut can le¿d 1o of the rlür€d DNA (Kim cl a]. rearrangernenls for the useof cornidsin genome 1992). Procedures in Evans(ttrisvolume) (see analysisafe described alsoEvansc1al. 1992).The losr¡idvccto¡ (Kim et al. 1992)conlaitlsccssitesfor corncdiatcd b¿c1eriopl)agc)" packagirg,bul Iikc BACS,il is derived I Jrr "r. 'ir gle.oo\ | ld'ru'u / .o/. .\L' I e fosmid vector ¿llows constructionol librariesbearirlg cosrnid sizedinsc¡]s that are rrore stablymain tai¡ed during propagalion than those of conv€n The insc.t size in cosDridand iosnid clon€s is limilcd by the conslrainrsof fáckagingthe DNA into rhc b¡cteriophagel, lhagc hcad. The Pl clonirg sylcm (Stemberg1990;Picrcccr al. 1992) packages(Loned DNA nr the larg€-rbactcriophage Pl phagehead,allowingclonedfragnentsto be as Iargeas 100ktr (seeSte berg,thisvohlmc).The Pl vectot ollcring the advan' vectoris á single-copy rageol inscrt srability.Small modificationsto tbe Pl vector.and a diflerent m€¡hod ol deliveringreco binant moleculcsinlo the host t. colicells (cleclrolorarion inread lrl trarrfection), 1edio thc devel' op¡nent of the PAC sysiem (IoaDnou et al. 1994) aDd the clonirg o[ DNA lragments hundrcds ol kilobascs in size. Similarly, rhe BAC systenl (Shizuyaet al. 1992), which uses an F lactor(ic¡ivcd vector has becn used to corfruct librárics aslargeas 300 DNA lr¿gments of statrlypropagat€d kb.

AVERAGE INSERTSIZEAND REPRESENTATION OF THE GENOIV]E In many gcnomic cloning stralcgies,DNA so rces and preparationscan b€ ma]]ipdated to cnri.h lor speciiicrcgions or targcls ol interest-Howevet for

Systems 1 r B¡cterialCtoning Chapter

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Construction of Bacter;al Gercm¡cLibra es libraries intended for genome'wide studies. the acrossa rcgion, and (l) províde evidenceol clone , \ p f u l n F .^. f I r e l i b r ¿ r yd e r " . r , l .^ r n ¿ \ i m r / i r . E lidelity to the lenome. The extent ol ovcrlap 'between clores in a library increasesas the size o{ rhe proporlionol ihe enliregenor¡epr€sentin the gcnor¡rc givcn libr¡ry. Fo¡ a lhe library increases.Altho gh there are advaDol a size,tbis is deterjnsefi (l) tágesto using large librariesin genome analysis min€d by two characteristics: the size ol jn (2) proiects, cloncs ihe libraryand there ar€ alsopracticaland economiclimth€ numbe¡ol clones ' I r l . l f . J r y t r r . r \. ^ n r ' i r i r - . 1 , , . , g. . r ' n i , its to their production, storage.ard manipulatioD (seebelow). librariesare usually charactedzedby th€ sizeot thc ailove,holv library.i.e., ihe nLnnberof cloneswithin the library Consideri¡rg thc issucsdiscnssed ¡trd lllc ¡nc¿ninscrlsiTeol theseclones. largedocs¿ iibrarynecd to bc?Thc 10!alamountol genomicDNA presentin the populationof clones Insertsizeis of crucialinportancein gcno¡ne an¡lysis bcc¡use il conlribures 1(l the overall in a libraryis urr¿lly severáltimeshigherthan the genomecoverageof rhe Library(se€below) and is a ¡mount ol DNA in the genomeof int€rest.The crilicalparamelerto the overallsuccess of the prora(ioof the amountol genomicDNA in the library Il the goalis to spaDa to thc ariounl to DNA in thc gcnome is reler¡ed to ccdurcsin ltenomcanalysis. as the nu ber o{ genomeequivalents,the depth ol large regiül where markers are availableal a mean genomecoverage,or ¡he genome¡edundancy.This sfacing of 1 per 100 kb, clores with a mean insert is ofren abbreviatedas lx or "l hit," *'hich rclefs lo siz€ of 50 kb will rar€ly link two markefs, ánd an al¡emative method for obtahing overlapping one genome equivalent. The calculatlon of a library'sgenome cove¡ag€involves lour lactorsr(l ) clmes, such as lingerprinring or walking, will be - \i/e r l , c r n . d r i n \ , the genone sizeofthe organjsm,(2) ihe total nümO r l l r e u r h e r h , n d i f "quired. ber oI cloncsin thc library, (l) lhe nümber ol llonof the lilriary is 150 kb, scrccningwilh rnarkersat contributing cloDes(i.e., empry wclls, vcclor only d n F " r t , ¿ , i n so f I p e r l 0 0 l b d l ¡ q ! \ u m c c S o n s clones) withú the library and (4) thc averagc Io be isolated as contiguous ov€rlapping clones. ins€rr size of üe cortdbu¡ing cloncs.Thc lormula sinlilarly, ihe isolation of a g€nomic region from a for determiring gerome coverage(c) is c = (total library with largeinsertsis accomplishedby scrccnnumber of clones- noncontributingclones)x aver' ing, isolaling,and analyziDglewer clones,simplifyage inseft sizelhaploidgenoine sizc. ing the genefal togisril:s o{ ihe projcct- Larger The coveragevalue indicates,on avcfage,how inserts may be impofiant to cover regions with many clones containing a parlicular locus will bc ünusuals€qLiences containinga low frequercyol p¡escnl.However,a genorn€coveraSevalue greater restrictior sites. Howcvcr, thcre m¿y be cases Nhcrc incrcasedinsert size representsan impcdirhan I does not meaD that the cntirc gcnome is presenl jn lhc library Instead,certain regiors wiI ment, e.g., the assemblyol a ¡andom rhotgun be present less olien (or cven absenl) and other sequenceol a clone severalhrudr€ds ol kilobases regionswil be presenrnore oltcn than the average i¡r sizc coLrldpresent problems lor the asñütbly ¡umber suggested by the cove¡age valuc. ^ssuming a random cloning of geDomicDNA irag Th€ size of the library is similarly important. ments, the probabilily ot having á particular Thc goal ot lib.ary confruction is lo maxlmize the genomic locus present in lltc library {ollows a 1ik€ljhood ¿t leasl one clone, and usually of li¡ding probPoissondisrriburion.Tablc2 shows the probabilities a y Iocüs oi interesi. The multipleclones,Io¡ of findiDg a locús wjilin a library Io¡ librarieswith ¿bility(P) of isolaLing clonesfor any regionoi the (see genome covc¡agcsi¡om 0.5 1o 10, assumingüat increases as the library size increascs ee oÍre . theg€nomc sizc is rnore lhan lo0 times large¡ than ¡elow). Furlherrnore,in genome analysis,a major Soil of screc¡inglibrariesis to isolai€a sericsol Theseprobabiüties(sccTable2) are an overes .o¡rligüous overlapping clones spanning large timatior duc to úe assrrm ion of random clorlillg, .egions. Iusl as the n nber of clones that c¿n be i.e., all of the genomicrcglonsa¡e equally clonable. rsolatedrep¡cscnling a single locus ircreaseswjlh pattr ln reality, some regions are easily cloncd, whe¡eas lcngth ot lhe continuous :he library size, lirc other regions are dilliculr or impossibleto .lone. r¡¿de up of overlappingclonesthat can be generalAlthough the¡e are numerous lactors that conwith library cd incrcascs sizeto is tribule to the nonfandom bias ol libra¡ies loward Characterizirgclores with ¡espcct ove¡lap (1) cefain gcnomic regions, two are mosl no¡able. :rsedro orient cloneswith respectto cach olhet 'lr allow the determination of paths of clones First, the restriction enzyme r€cogniiion sitestnat

Chapter1 r

BacterialCloningSyslems

Table2 Probabiliq ol Having One or More Clones/ LocLrswithin a Library as a Functior ol Library Size

f ¿ b l e3 \ J ' 1 b . ' ñ a l o n ( \ R e q J i . d l o r 7 1 >

Genomic coverage Eaploid

l9.l 63.2 86.4 95.0 98.2 99.i 99.75 99.91

0.t tl

2 ) 4 5 7

99.99 99.995

9 l0

js Th¡ probabiliryol lindnrg ¡ clonesI¡on ¿ litr¡¡rv ol. Loverage P(r) = =

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TIretrobabihies shown a¡e ihos. olli¡di¡g o¡e o¡ m're rl¡nes lo Ir P(o)l Jo¡ librrries wilL gclome.olcmges I.om 0 t lo

are used for cloning are rot unifolrlrly distribLrte.i in geDomicDNA. Second,rhere a¡e regionswithin g e n u m r\ce q u e n \ c r\ l r " .d ( c l h ¿ lo r p u Ú r l )r ' ' d i n tained in bacte¡ialhost cells lor thesereasons,]t ts usually necessary!o conslruct librarieslo a greater d e p , nl l ' r r . , r g B < n e L d ' v . r ' eP o i ' 5 u ¡nf t r l i , i ^ n genome coveragerequired for a The depth oi genomic libtary depends on tbe purpose of the library. As seenin Table2, it is very likely rhÁ1a 5x library would be adcquatefor linding one of more clonesfor a locus of inleref, sincemore than 99yo ot lhe genomeshor d be present.ln a proje{r where only a few clonesper locLrsare needed, lhe addi' tional reagenlsand eflor! required io co¡srrrr't a l " r g . l 0 ' , . r " - " r v a r L , . ^ $ o r r r n l e dH o { e \ r r ' I walking or mapping,where eachclone h a seÍes ol clonesmust be identificd, ¡he chanceof fiiding no clonesat somepoint in the parh is grealer(beingrhe product ()1the probabilitiesol finding eac! ol ¡¡e clores). Thüs, on a shtistical basis,e g., when con' slructing clone contigslrom a 5x BAC library, gaps are expected,on average,every l0 Mb. Libradesot greaterdepth (>l0x) not oniY provide an in'reased . i k e l ' l r o ou d l h u i l d ' n g( " r ' i n r ' u . r \! o v c r c a co v ( long paths, bul also allow a flexibility in selecting specificpaths (e.g.,a path of minimaUy overlapping clonestor sequencing).Thus, lhe appropriatenumber of clones in a librarY should be the minirüal number ol clonesgiving a genome coveragelikcly to meel the necessaryreqldrements

no. ol (Mb)

(kb)

20 1000 1000 1000

50 t00 50 150

1000 7504 450,000 150,000

no. of 384 weu

a 20 t172 l9l

CoDstruclingand scree¡ing highly redundarl librariesfor large genornesrequires generatingand handling many clones.This increasesthe cost and elfort for dreir production, storage,and manipula_ lion. Table I sumlnarizes the nu¡tbers of clor,es required to prod ce 7.5x coverage libraries for organismsof dilferent geiome sizes Libraries tor organisms with smaller genomes requjre sm¿ll numbers of clones. Fo¡ inslance, a 7 5x fosm;d library for an organism with a 20 Mb genome cor d be slored in a set ol cight 184-well mjcrotier pLa¡es. A 7.5x human (ge¡ornesizeof 1000 Mbl BAC librarywirh an avcrageinscn sizeof 150 kb would occupy hündreds ol mjcrodter plales; a 7'5x h man cosmid library woukt occupy more than 1000plates.

DNASOURCE A genomiclibrary relleclslhe genomeholn which iI was made. The selectionof parlicular individuals. s¡rains,or cell lines as a DNA source can cnh¿nce of Lheresullnrgübraries For instance charactcristics adiac€ntto a specifictrando Lhe sequerces io clo¡e iglt choosero constru( Ihe one cationbreakpoint, of lhe indlvidual wilh that DNA lrom ihe library in üc context oI geDome Howevet breakpoini. is most olten chosenlor ill DNA source ¡he analysis, configuratior of the of the "normai" representati('r genome,rathcr lhan for the presenceor absenceof specilicattributes.Even ilr lhe laiter case,imporlanl issuesrnusr be considered,e.g., the use ot tlaJls lbrmed cell lines. If a cell line has beeDex¡ensivelv d r r r ¿i ñ n . p u ' " 8 e d .i r i \ l r \ e l v . l , d t c " . J n , u l ¿ r em "nomalfuom lhe that deviate and rearrangemen¡s passage cell an early genome, and lhus stareol that orga¡ The sex of rhe choice line would be a betler ism that will sewe as the DNA sourcemay alsobe a factor.In malnmals,twice as many cloncs must be

i

GenomicL¡braries Construction of Bacterial

s.re(ncd tor X chrornosoneloci whcn DNA tron a malc is used.comparedto rvhen DNA lrom a iemálc is used. Y chromosomeloci will nol be preseni in DN^ from fenrales.If it is likely üat clonesmay be used larer ir hrnctionál knockout or ho¡nologous reconbina¡ion experiments.there ay be advar tag€sro using DNA derived from the same animal rrain as the recipienl ccll. For the human genome, ¡¿tLrral variation becomessignilicant.To obtain a library that minimizesvaiation, DNA lrorn a con_ individualcouldbe used;for a spccilic sangLrineous clllomoso €, the chromosomecould be flow-sorled Irom a monoclrromosomalsomaiic ccll hybrid on (hc olhe¡ hand, to maxinri?ethe varialionsl}lai carr b€ identilicd lrom the clones in lhe libfary, DNA IÍol¡ multiple individuals could be pooled. Finally, erhicalaDd legal issuesnlust alsobe considered.For guidelúeslrave large scal€humán DNA sequenci¡\g, proteclion oi DNA hccn issuedlor rec.üi|ment and guidelines, secthe donors For iüformation on these Rescarch/ ^"alional Cc¡rle¡ for Hulllan Genome Guidanceon Dcpalment ol Energy (NCÉlGR-DOE) Hu¡nan Subjects Issucs in LargeScalc DNA at ihe following World Wide Web ScqLrcncing / http: / /\\Ñ. ornL. qowe/TectiResou.ces H'¡nan Genone/arctrive/nchgrdoe hLml

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each fep, it is important to be able to oblain large amounts oi DNA initially or to be able 1()repeatthe DN^ preparalion as needed. Diifcrcnt bacterial cloning vectors can acceptdifferent size ra¡rgesol geüomic inserr DNA| fiom 15 -45 kb for fosmid/ This cosnids ro as much as 300 kb {or BACS/PACS. has irnpoftanr implications for the preparation ol the source DNA. In gene¡al, the average size oI hagments lrom the p dlied sou¡ce DNA mrsi be five to six timcs larger tiran the size ol lragments ¡equired in the ligation slcp. In particula¡, thc preparation oi DNA for the construction ol B^C and PAC libraries uses source DNAS isolated in agaroseplugs (Shizuyaet al. 1992;loanno et a]. 1994;I
THE BACTERIALHOSTSTRAIN AND QUALITYOF DNA REQUIRED AI\¡OUNT The quantity ol DNA neededto constr cl a library f.ill va¡y depe¡dingon th€ cloningsystemchosen rnd üe extent oi genomecovcragcneeded(see T¡blc 4). As library consl¡uctionofien inl,olvess€v_ r:¿l rounds ol DNA preparation,size-selection,lig ¿iion. and lranslollnalion, wilh optínization oi

CeÍain DNA sequencesmay be dillicull to clone and/or rnaintain in a bacterial host. Many E. rrli murations have becrr identilied üat improve ihe abiljty o{ a strain to scrveas a host io¡ ioreign DNA r i g l rl e ' e l "o f r F t p . i r i ! -\ . l u c r . e \ i n T.r iIrrcf genomes ol higher organismsmears that mollhe ible copies of thcsc repetitive sequenceswili be

r¿bre4 clonins Merhodssuiiablcior GeneralinsG€nomicLibrariesu.i"s¡l!{l!!9llg{4 llife¡enrf ref¿rat'ons

(pg)

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:-¡nomic DN^ nr aqlrcous

100 200kb

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-:.n rmic DN^ in ¿queous

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systenrs Cloning 1 | Bacterial Cháp16r Dresentwltrin individüal clones' Wild-tvpe E c¡rl¡ strains are highly proficien! in recombiDalion' which causes dele!ions a¡d rearrangemenls rn case through Í]lramolecr¡lar ¡ecombilration(and recomblna oL mutticopy veclors, inlermolecular Poor tion as witl¡ a¡ lhese repeated sequences oi lhe cio,rabilitv aúo resul$ lrom irrcoñpalibilily and reslridion Ioreien DNA with rhe natural host modiflcallon sysremsthe choi.c of host strain is an imDorta¡l factor lor the elficieBcyoi lranüe'lron or r.airstormaLlon,and the quantity a¡d qualily oi DNA obtai¡e¿lfrom cultures To rcduce the ability

ol !h€ bacterial host !o modily cloned DNA fragmcnts in such ways, scveral host strains wilh m J r r r . e e n o l v p e¿ sre n"w ¡r¿rl¡o'' or libran ¿n'j L o ' l \ r r r . : o n L . e el ¿ b l . '- ' l r r L i c n r r ¿ lD H 5 & llHl0B are uselulfor cosmidlibraries,and DHIoB has beer used lor BAc and tosmid library con_ rhe rvell struclio¡. Table 6 slr¡nmarizessome ol known E. ¿rlt gencs that are directly or indiredlt ano involved with DNA recombination processes host restricrion and modilicaiiotl, or otherwis€ inlhrencethe sulability ol a slrain lo serveas a hor lor clorcd liagments

of BacterialGenomicLib¡aries Const¡uction

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Systems Cloning 1 ¡ Bacterial Chapler

Table 6 E. colíMulalions That Allect lhe Ability to Clone Marnmalian lunc¡ions and mutalio¡al effectsof lhe genc

DNA in Dillerent Hof Strains

increascsthe rabili¡v oI DNA rhár would The major gcne involv€d in homologous.ecou inatioo MlrralioD repc¡itivescqueDces (e'8 containing DNA ' ur¡e.*i'" üe p-"e rn delerionsot rcarransclüenrs Mutatio¡ inoeáscs the rabilirv ThesegcnesencodeexonucleaseV and are also involved in ¡ecomtrination oiDNAthatwoul.lo¡herwisebe|ronetodeledonsorlcarfangcmenls(e's.,DNAcontatnnrgrepctLltve 's reccss'v tor üe '¡pÉsioñ of rhe The ge¡e eD.o.tts a componc¡! ol the RccBC cDzvmccomplcx and DNA that would otherwise bc proDe ro cnzy;et nüclcolylic adivi¡ies MütatioD iticreasesthe stabiiitv ol ilelerionsor rcani¡eemenrs (e.g, DNA conlaining repcdLivesc{luenccs)' The r''¡ The gene enco.lcsá componcnrol dre ru'¡ conbi¡átion pa¡hwav

mutalÚD i¡nprovesrhe growln

I, a co¡iponc¡L ol rbe ru'FPaLhwavThe J¡c¡ nintario¡ tr¡provcs tlre erown The genc encodcsexoDuclease ol r¿daaDd /e.c nlurams methvlareDNA but are unable Thc geneencodesa sul'uDitoI lhc ¡es¡rictio¡ enzvüreEc"( 'rdR ¡¡uranls can lo ¡estric!lorcigü DNA a¡ rbc apfropriarc recognition5i1c a'le¡i¡e rc:idúsin,Eú( rslricl'on The gene encodes¡ subunit oI E .ol; ¡ierhvl¿se which mtllylales lhe residues DNA rr'm ¡n ¡J¿'ll siles:Thc¡t ¡(¡crricrion e¡zvDie lalls to recognizeDNA ¡¡clLvlaled al adenine . ' l e d i- c '-\dt l'o\' r o u r a n. l r - r ¡ * r l " e r e l and HsdR protcins /rr¿i Thc ge¡e eDcodesa lrorei¡ subunit thai co¡lers sile spe'ilicirv lo boü HstlM ¡rutan$ are in.apable oI both melhyladon and rcslridún ntthylaLed al Ihe scquedce The gcne eDcodesa ¡eslricrion enzyme. Muiation prevenis resl¡iclion oI DNA mclhvlcvrosi¡e rcsjd"es 5--C;"CGG-3',thereby allowing morc elljcient.lonlng oI DNA conraining Mutation Dre The n¡:ra and mdc Senescocodethe ¡wo subunits ol the McrBC res¡ric¡ionendonucleasr' cl ringolDNA cllicient ¡rue thus allows and 5-_GDcc_lscquencc ar lne ai melbylcytosúc vcnls resrriction con¡aining5 mcihylcyrosineor 5}vdroxvnreúYl cvlosine' a¡ the sequences The Sencencoitesa refricrion enzylne Mu|alion Prevorts rerri'tion ol DNA ¡tethylated nrelhyladen¡ne coirainiDg of DNA :-_ci.46-r- n. 5--c-"Ac_l-. rhcreby atlowing mo.e ellicienr .lonilrg a¡d has ürcreas'd The genc en.odcs en.lonucleáseI DNA pu¡itied l¡o'n ¿'¡J¿mltaDts is of a hjghe' vleld s!ábilily due to thc absenceoI üis nuclcasc raDdonna The genee¡codesrhe rc!ressor ol thc /d¡ opcron. A mut¿tion in rhis genr aLlowskr irrc'caserl tion rlticieDcyol largeplasmids lacz Ml5

thaLlacks amino ¡cids l l 4l Some clonins This .lcleteclsene enco{lesan ináctive tom ol p'galactosidase vec¡orscncodeanaminotcrmlnalf'Ifag¡¡eDtoIpgala.losidasc'Thisallo]vs(,comple¡¡eDtaljonwiúüe hosr/4.ZAMl5produc!¡olormanactiveenzyme.Thcclonilrssile(s)ofsuchvedols]ieswithinüedfrag. ment'e¡codnrgregjonTherefore,no¡recombinantslortnbluecolonicsonmediumco¡rainingxgal whercasreconbina¡ts are wbüe

^^crA, mdtsC, and mn all rcsrict DNA modilied by CpG ncrhvLase

Bacterial Cloning Systems

DNA I brar e; uslng E coll hosts. fhe ease of application, coning capaciv, a¡d re jability oi baclerial cloning .... using baclerial cLoDes. second, thc need lo analyzc ..... This increases the cost and ... ScqLrcncing at ihe following World Wide Web.

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