a a FE. Ne se racti rman Maree ee serecp in oa sgenic mice. TNA can be injec tates ‘ Miigetienoeeretal silo ee crane : ~at Gene Cloning Strategies aa ‘iad : Ee 2! te ce tee re ed a cutinaprcerepeemcnn, nib nee ey eect eee Sangeeta ae eee eae anne ps chai nan spi tense tres Comparison af Genomic Liber Rett meta eck otwchocs wih Book Library “Siecictnc secvormavncnes “pants et yoke {foe DNA isolation from source organism, f0¢ pared to the classical book Ubrary. in a book ‘cutting the DNA into small pices, fr join- Viprary, books are stored while in the DNA ing of DNA _ segments for the transfer of jibrary, DNA fragment are stored. A book is DNA ito hosts and forthe selection of fe- safe guarded by i cover while the DNA is ‘combinans, The choice of techniques wo be Se guarded by the vector DNA. A book is {toned dents ent muerte abn sl oles DNA ag Inge meal esting mi jonne rel insbnaea acs ek May eva Sisal asboak lvls Asetoftechigues, opi Son Classical Library DNA Library 2 gon in opm toe gee " 4 Ung rag. Tose we cel ees Book , Seen nny doping o wr We tesco gts eof DNA iarsateicer seman! ]fsoyans ©) peceeteDNAcrapatinirpeceatte | [fh bound BNA Seyret ave tsn ae DNA stuched to 1. Getomie DNA Wary es 2.eDNA hibary 3 Chromosome walking 4, Chromosome jumping Bookshelf =f 41. Genomic DNA Library mae ‘cer A collection of eloes containing all DNA segment ofthe genone ofan organism Fig.1S.. is called genomic DNA Iran Its also Comparison of boo Iirary ik DNA brary202 aa onicaveas ‘turing the host ell. mt Ht ne eine Sioa Hisoas beretouracn Sem ec eee Scheie igct ee oe care ‘ered Geen tan han arc ee awe nd Es eB SST pmlc See fn oeanicalimhctatete Ps oraienterirar rian Bek ee ata as Sancta ier sat traces at Sane Sen dctutinatne see irae chee SOAS AS an te mene sco ele te Seen ee EAiigpoaste tay ea recon mone sa ow toed (ty eg sro Cae cue fares Car mae obo tc. ober ae te omc 2 ng Dd Serica eben Te ekind eceeaue oe eae kate relate Tees rm ges se rs lege a Telos na FAC iy rr 065 ‘Satish nome ecm human genome Wiehe nai ay. ny $0,000 cer coe sted Is lone in VAC ny 10000 loreal ough amo Creation of Genomie DNA Library ‘Shotgun cloning method i wily opted terete pene DNA les oie a DNA fens fa genes ‘Soaved vector ta fine by esa eg periment led shou lng Toe Totlowing steps are et fo come! a pe same DNA ary 1. The gevomie DNA i led foo, the soiree ogi ig sade pce ue 2. The aad genic DNA sca a resatonenzyne get all DNA ftagments- While cating the DNA wie ingle restriction enero, loner gms tay be generated Wn, random et a Irae wih two diferent esc cozy ‘Tis thd is ald Monit rats. 13. The estition dpe s econo sed oma polyacrylamide bln he preset fof molecular weight ares mor ne “t DNA feaments of arable si a isolated rm the gel ad the salle a ell 2 larger feagments are discarded. Fore Spl ifthe DNA fagmens ave tote ‘toned in plasmids, size of he fragment Should be between Sad 13; 0 apmens ese than’ band agerthan 15 Roar di corded 5 "The isolated DNA fragments ae treated wit aline phosphatase to remare 'sphosphite groups from the end of t= DNAS (6. The vector DNA is cleaved with one ‘orto estiction enzymes which were we tthe genome DNA, "The dephosphuxylted DNA fags ave mined with he linearized vecar DNA 0 ‘onset DNAS. (1.15 SC Core ss 2 ina “ 00006 000909 Phsmide louse ‘st gO OOO zz, VOOUU Lares, oa6ao 60080" DNAS L “rasfomaton in Ecol kayo nmi’ ‘DNA shawing ‘ind sie 1 Selzeon of combina Recombiaat Eco Screg by inno ‘hemical met -— TRRERLESEY TBs ones [Geno iy ig 13.2: Contraction of Genie DNA ibrar in Ecol sing lol wo 8, The #DNAS ths constructed aia uodcdint Ecol oryenstel. WtbeDNA Fscloned nla, thy are inode into Eeoiby meansoftanstormation ItbeDNA. fs clooed in cosmids, A-DNA cr asi Trnsdoing vis piss ar eloped 19 DNA a yea cel iver he DNAS imo Eel. DNAS hood TPAC and BAC redler Ely ‘rarsormaion vig eetporaion 1 DDNA bs clood in YAC, tafe wh fete eltpratinn is sa deer BSN08 The cnn weed by ‘ac approprin mtd ifr esl or e 10. The eons ae sept Som oe ater wing on pst sbsegunt etre 1. The mri clare screen to det he cong 12. Based on the cams the ‘tones are aranged inte peo lea), Screening.of DNA Library “The clones ae aang nto contigs eatesclon terns fovea eee sf acentclones Reston ag nrifine erriming chromosome wang eine DNA fingerprinting and STS eauence tages apg ont sons ones to cot In chromosome waling the genase DNA fs et wh ander rican eye to gneate ovetping en The et mens are aed wh lative poses to prapare radi por BNA {il he cones are ltl ons proctoe Titer anda poe atone toyz with ‘hem. The postivey reaiag cones are ‘ought ogee as aeent clones. Ths the clones are Brought tpt eny- ene ino asec of ootnueas DNA dns of ‘nism. Te comin cones resent Ing the entire somone DNA of ngs the core order eld comin Inrsictionensymefngeprnang be svomic DNA ica wih wor more ete tn enzymes to gt define hane of DNAS ‘Theclonersre asc wi heey sp ‘ely. Twoclonr hat pode sete tons Wil be pene eticon eye finger rin are Bough tpt rjc clone. Restriction aye fgeriing ante ana "yz ony wih modem corps Inrpetive DNA ferrin. sce tive sequences are lowed hybrid ith DNA ized fom the cons. Te eps {ly easing donee gh gsr cot clos ‘Semone SS) aig ‘egoretsond naepmme se 10.0 tp bog anes twee ‘ating nes Tse sage STs on ret on oo aces Dk spoons Ist aren gn ess se deted with PCR an eel cee Ciccone ing esa ‘Afr arnging al eco «proper ict eit ren gens, thesonesarcaulopsl and pce th omic DNA iy Frobabity of DNA Fragments To comets grae DNA ey DNA grenoble fm he ene DNA ofr ees Ye ce as the host oe. Bat by pons ‘etme some DNA fgets af Bos Secloning precede Besos een ‘eincsn afsome DNA fap seen ties de othe resent of or ta oe {py ath fens interest ge ‘htt th ber do gil n the ary ust be bays per a tmnberofDNA apne ga the gownie DNA. Tn an al genome Ray ll DNA Sragnets ms ave eres wh ep rroabiiy The minimum ser cons Tegel scommodae DNA fens {af genomic DNA can be decried oy ings rpc Crean Ca ton in 16 Nis he mumber of clos agus 0 represent the genomie ary P inthe expected prbabiiy ‘inthe number of DNA fagneats gs crated (genome size divided by reo inert DNA. CHS Go Cesbe Sens 2s ‘The pohly of DNA faparble_ Pamah we car RDN ‘be nomi DNA Satay 10M cancury 15 tong DNA ropes Re eteptabiiyis9S% tease pine apne feet WO ace ‘ost the DNA fgnestfom te ayetche isSce DN {i igh pots acieed 9 reptine sere conte es ‘ate DNA ay ‘mit eDSAe laying ean cn Uses of Genomle Library ine pee |. Genome ais oie DNA fag" Lamberts verti ile sens of eens fer suing poe DNAS th can ey 5 ng DA Ty 2.Geteofitreten broth em an be ely poppin Bel The ok ‘be gznomic DNA iy forge alse oi gh The ak ‘en wer 100% probly cn be cee 3. Genomic ire hp fr econ Phas can ary 20 ong DNA ston of plz map ofan. agmens The recombine gee an “CCintmc ihc eect say pen nye sietgncrtain sagntrome. BE aie Cesare pre 32 Ga sig heme NAT as cOR ey cahcemistymetyneines eal ile ek Sococks ri ‘reatono cONA Ubroy cDNA Liars ec of DNA ty ma Acohatoefnaectfither Witt Wise eta vaya te, Ss dao eDNede y fee penion a se apes be COMICON of cONAS [Seine paeettecean i gy ON ny nimcarssagmpos bone Qt caneamiuDN meal ‘eae ees tag esr ox in mRNA ‘Sects ste iDvA e , , ENA ati om ‘canstructa DNA liraryallcDNAspepared, "¥en Se Oe ae Sonate ionsetactaaedMeeASuade sta tos els ough isle esos. The ge sunt Di farmed pon CN Vectors Used in cDNA Library jrecseD elma TecONchoe sts) Since cDNAs hve ma ion, tiie Fr gene cing vata es thn 0K, Tetra The eDNA cles are pray oe vector at ean c0emo- lowing ep oer Scag DNA Geely pms Alton AWA ssa phagemid wed ast A ny NA rei ‘DNA aries Hee, cDNA Uso and uo Is blared nee ‘ante with Ecol cals206 2. Reverse Transcription “The fail of MRNAS in sftion is rte with go primer, he ene vee menserpe a forts deat rectvades The wee ned a SSC sie fe ert, “The oligo AT) pine ge Pond with pots ial ft hance MRNAS. The mer provides fee 08 up 1 p- Ered the eves ean Revers tania as complemen: tary devices ne by e010 the RNA, aay b WET ee Revie AAA ee ys Saicm sow ot a, nau y TT TITS : ae ’ hit 30 sia Alkaline sucrose solution 500 AVNET secs shee ing seen Tris SGG6G0I AMA «DNA clone Fig: Moing fl eng DSA clone from mRNA soreanexocr Tv i al oi Seoul DRA Asean BL Ima Tego Ecwonnay te esc Snponntry De CONN 3 bigo a () tating ‘ts ong NDNA tra withthe cnyne terminal rae Ste matended CTP. Terenas als CrP ove by oae fo he 1.0 propo sh {heRNASnUDNA sands Asana lige d (fell fore the ao ‘terran, Poly A Tail SITTTTS al CWS Gn Cun Sacs {& AtkalineHyarofysl of mANA "Te ation ite ttl with Aatine sure slain Te alae ‘easton epee Be RNA fl DNA stands and separate he 0 rss 5. Addin of Oligo (6) Primer ‘Oligo dG) piers ade ote ee, sonmixtneanteengente smite SSC. Thelin) pte ges eur ‘itholgn i of Be DNA ad RNA Dyintrchanbydopentonting The inet vies 3-01 grup pecs orm 6, Synthese of the Second ONA Sirand "To te acion wit, enw eae and all te ou pps derbenees fre asd. The ene scone Ilion ene he 3-08 Ben of Tepemer Arent saablsranelDNA ‘allel eDNA lonesome neh eDNA. Cloning of cONAS "The DNA lng invest fallow ing sep The cDNAs ts bie jd ith che spo wing FEDNA i. teh cDNA rhe etl wit Satine hosp cme 1 remove SP ‘0p fom the DNAS The yor DNA yr eg DNA. cut wiha rotten oxy. ML The eDNAs and veto DNA a6 nivel gather and teed wih DNA BE, oh cDNA gt eel Reem cot thease BNA, Thecossivincte DSA Stas wih co site of amie LDN ant fence conte ae fen. Hentai and ok pins a torte DNA intl Each ose ‘ieechage inhi ease APO {Soin vis ea ets he coon at ate of the prkagd strane. The ‘eek an alin with apne Te ‘no page pre Ts ny BE fue a fe te 0 ae 2/F od Ee oo Py hy 4 Eo Eel Est Ooo Hoty oO 4 it ing Sante oss ae Oe Giepes Ct rening oe eDNA ry Fig 1S: Stops imcolved in t consraston of DNA Hira.Nv : The recombinant pags pails are ag ineeta Bett cotae. «Then recombinant Ecol cells re se lected by using a suiabe metho, +h The Ecol eal contsining com binned ad suber to separate individual bacterial les, Screening of cDNA library Analysis of various clones arrange ‘them nto camigsiselledsereeing of DNA rary. This idan with reson encrme fingerprining, STS mapping and DNA fio Mepining The stg of ‘Sur DNA ihe oes Thr ep Cie erorccane wane Chrome wing inaes 0 - jorge infirsep, ctel arse ‘nel apn fs rome DNA me Comte nth aon sep he oi os los ae nie a ene Sil of oetaping ees. “To conn! hte lone, the nomi DNA isco sal pases eg ‘re cay and te DNA pos st cnet trv alls get In hese cloes the ea DNA open Indoiy dnited Wea ow ni Sfp cst he fet DNA fog a ‘homorome hich oe cntes et oad ‘Sov Ba sw hat treat ofcles © {ge hom in cops che, "TermonieDNA sons pn ibied frm he source organs wih ter ese nye rs doe Itnent The reste ae to. ordi parte DNA tapes tase {nti Safer ad grape ae {heme Matus DRA ame = 20 ky ae roll tn ett cp ‘ely DNA taken cath an ee and inset no DASH och The ‘eon cr pcagedia psd ‘npg to pode bia aera {len These pages oe london CHL: Goo Con Soames "Ee cell The renin eli ar 3e= ‘stl byw pl sels meta ‘The DASH a FX cota T3 and 7 promoter acetone ings. Therefors thecal DNA fag rns is ated in mRNA heii ls for ping. Suh pees ale end “Spec RNA probes, These rabes bt then sae eo ating stn ‘mesa aed the ttle. Tee ‘ten eyes ute clone gent ‘no sal segments excep ow mice ides taboo te pate See ‘pel RNA probes rains. Whe Toctne pple sd oe acter alte, eadoltelldendpeii robe is Syotbesved. Tha, several sch pees are ‘peped freeman waking (artigo) fer i Clone} ClweS Cnet 209 DNA fom al helene eked sen no at dots on tele ie ‘Theirs ey dip in slton cent {ng one pce. The pstey ecg lane Ssclosed suring clone The tii thong with he eon dpb, forth robe snd soon Repeed yi one dtc oe clone everytime tt Fah ofthe previous elne. Av esl eve cloner a range iat a 30 Com tics lone Uses of Comosome Walking 1. Comore waking ised io Inte unknown sequences found px 0 Krown ere 2. Genomic and cDNA lieve are screed with ehumorme walling fang tbe conse cones io comes or fstlogung the clos. Gone Clone? F153: Sups imo crumsome ali