Chapter Outline Chapter Outline = a ad rae Chapter Outline “etalny fumeton | _apention “Galacrophrs—Tosnpuate apni of cant teint 2 MeptcetndarONA btackerdngtolngh | Dakine 2 robe mn Poet tne era raeecingae Sn Stinging {Alcormatis ‘alleen proceing _* fot in dog development POulsequncne sani flees Egor ames (Gen ot rene) ilo ete ving“fone: ene ty MA “+ Amplify = production of many copies of a length of ONA z “Cosma mg rtd Polymerase Chain Reaction (PCR) “Rap article process @r (4 Components needed ~~ = Sem nn 2 mapormnae en Option hematin ae 4 Sopmatmtroanin co) =f i 3 amen oun 26s j-- Samoa -epenmarrsea pt otal ont Polymerase Chain Reaction (PCR) f 2. maton 5 “OWA stands separate / denature nto 2 TTT ‘rand by hast + hdrogen bonds between ONASttanés breaks] mmm ey Haat pried wen = + Produce template strands or oping Masaauan ee Thane 1 seen ang rae eT Polymerase Chain Reaction (PCR) 2. Annealing (6065°0) “Primers anneal bind wo specie section of DNA + Via complementary base paling “New hydrogen bons fom fate of primes + indo target ion for ampiftion ‘+ Actsasa stating point ofr Tag poymeraseto bind [mmm tty Toa polymerase onl bids to doublestrande4 DNA a 3rd odd new nceoies toon eng rah ——, =a aes im Polymerase Chain Reaction (PCR) extension 705°C) + Tog polymerase nds to primer ee + Sythe: new DNA strands + complemenarrtotheNAtempltesvands TENEETEET | ae ‘Tog polymerase has a high optimal temperature SERNNT TSE [ikea he ertna Aassaue + Doesnt need replacing each cycle 1 rT 1 DT = n Polymerase Chain Reaction (PCR) \ + ons heated again sept stands + The proces rapeted unl sficnt ONA produced (sly 20. 1 The numberof ONA molecules doubles very eye = effet proces! + Number of ONA double hal opis made rom ‘ne starting molecule ater ney of PCR > Polymerase Chain Reaction (PCR) i sag: ene {Duron onl OMA ede mpc OMA Dladvantages: + Need to now the pedis DNA Sequence belorehand to design primers ‘acer (GenetTeehnue 2) Applications of Polymerase Chain Reaction | K Fnetion: cane and amply DNA Appleton: + DNA sequencing > tole to amply small amount of DNA extracted fr sequeneng (Eg infos blod sample) + DNA potting > Able to amply small amounts of DNA extracted (Eg crime scene) + Recombinant DNA Techlony > Amplty DNA/gene needs onsen nt pasmid + Ganetiescrening “> Toidenty maatins/iseose enes/ONA tom pathogens Use primers complementary to aa ene ‘> Toidetty and amply trget gene in specie 3 Gol elcrophorei uted alate geneenetic Technology 2 RECOMBINANT DNA TECHNOLOGY / GENE CLONING Function: to make many copies of one gene / protein, Recombinant DNA Technology | SEs eur f tm) TN eaten ; : inelin gene wh plasms = > tenn me 4, Insert recombinant plasmid into bac, j IM 1. Recombinant asmis mised wt ace 3. top hes shock 420) rae lactoporton to increne chances of ‘nas pecsng trough el surace membrane 5. Identify modified / transformed bact Yemtireane wing lad Exrpaumajatreamedwn tit Ue anit tlction ‘Urn graen florescent protein (GFP) ) Usman esiy stained subetance ( slactoidne, GUS) | + Eggenes tor anti resistance genes for Muoresent/ ‘uy sine sbetances {ath target gene and mae gene re expesed + FP arts riht ren ht + When exposed to UV + Sacer with pani wil xpress GP and wit a) Antibiotic Selection Emtec gee amt resttnc gre et + row on agar containing te ana (msicin) {actra with lai witb ale srive ne. ec + Then mate aap plate by wing spenge/ele pad p + Grow bacteria on agar containing ™antbiote atraqetine) bt yaa {i a) Antibiotic Selection f vor rose | 1 lek of spread of antibiotic resistance to ther bacteria of ame at Ql + Paani are ely wanterred between bacteria a conjugation + Slower process for denesion f wanformad bate swell * soe other methods to nty modified bacteria b) Use Green Fluorescent Protein (GF; ji gener Green fuoescent rte (1?) “+ Use gine that codes for eal stane abetanes ae marker ¢) Use an easily stained substance , E me i Ee ener Bgaacolsae sa oe one + rayne spits a speci nan ue substrate it produc that ue acter with pari willbecome ue6. Grow bacteria in fermenters for large-scale production ‘ow vansormed bce clo muh /lone ‘row in arg-scale cute ermenter colcan dvds once every nine ‘Sacer produces multiple copes ofthe gne / protain product (in thease, uma nin). Sacer nas elation, ansrintion and rtaton machinery to cpy the gene and express rotsn product 1 ONK polymerase, NA plymerse, rBocomes) Advantages of Recombinant DNA Technol: [Advantages of producing han inelnby gone technology: 1. Chemical identi to human sulin ext to nsuln receptors on age organs 3 doesnot simulate the immune sytem S tse no nk allege reaction 2. Effective in people who have developed tolerance to anima dered insulin 3. Avoid ethical issues related to reliion use of animal roduts rotilingof enna 4 Lower cost of purifeaton and processing 5. Mas production = larg ad constant lable suppl yea ound 6. Laer of contaminaton/nfection rorihof waster of daease 7. Potental oengner/ improve recombinant protein Applications of Recombinant DNA Technol E + oducon of pharmacats > Nomodying of protein in bacteria (bs no membrane Bound egal > Coney mat etaraarisnmen pes ep bod detinhsenepha Gl hater ey andy pens ‘aleiniomete prin Enohyens Mato + Usedingeneticanhneerng to produce GMOr in greGenetic Engineering vam oes of ees. + ofthe same ora species + Hove recombinant DNA rDNA) = combination of DNA rom te or mare sources + apressesthe new gene product protein + Genetiay modified (6M) / q GMOs in Agriculture { f ‘Aims of using genetic engineering to produce GMOs in agriculture: , Tinto urn ocop par oath + Incest op pas nd esesh URS SS irs Ee RNS GMOs in Agriculture 2 are eh owen te + winner + ne ger praca be engere te: +The er yl eer guy ae Dl pe peeves + Glow nations ontons/ mare talent clmate hoe + row poo quty nd ee eee “> aortas ath bene teste food chai plinaton GMOs in Agriculture Why we GMOs insted of artical selection? + Much tsar et GMOs in Agriculture Important example: + amin Aenhaced ie Golden ee™) food reduction GM Atlantic Salmon + Normal satmon neds 3236 months * Genser nom tps sn “+ Genes for premoter rom another it species Bonet: + High yd dl + Allmodifid salmon eggs are triploid and sterile > so1mpossble for them to breed among themselves and with ater sl 2 eliminate impacto wi population &Golden Rice™ + Problem: tami A deflency in developing foumtries > binds + Ree is3 staple food forms malo part of et + amin found in aleurone Ine ote eed, + Alurone yer removeatrom whe ie during arotane inthe andonprm | ler, + Beaoteet meabotsedin te human body%6 2Linsert genes ito Tp + Using restion eneymes and OMA ase > Form combinant pasid Golden Rice™ 1 precipi scr Arter mec innenmn he + The oNAinits Ti psmidisvanstened moos pat and isinsened me tmeteconaae 44. A.tumefocens mised wihice embryos ~ + Some embryos tate up bacteria Nw vitamin A gane enters embyo cle == ‘soon Lule Golden Rice™ Advotags + ete quay ood Disadvantages {+ May ot rom wel inal conditions a other ats nt selected or + Gat seedcoudbe tut fr frmersindevaping countries 0 btn + Farmers canet ue own sed + Might ceduce eos to raleve power whch isthe mame Insect-Resistant Crops Bt Crops x Bteoton, Btmalee + comborer eats leaver nd brows nto tak “ant cant suport the asf com ‘hringensiinserted to maize ton Insect-Resistant Crops Bt Crops = Iereated yields + Only il specie insects that eats and foes not kibenel insect Et polinators bees / predator of pests > conserve bodversty ood web + taped ued “Reduce is of peste atletng other non target species inthe same + Les f harm tohumans from spay dit pestle residues on Herbicide-Resistant Crops Glyphosate-rsistant crops tobe end pe, ound andy seyean + cenetay mode's be tnt wheres coting Shonen (ee Rows + Gen rom bacterium Agrobacterium coding for enzymes with sme fnction hat ae no ected by pphosate + Herbicide resstan gone ats 2s mater gene 1 Herbicides wl hve noc on plant, oly the weeds YO Vy Herbicide-Resistant Crops Glyphosate-resistant crops Advantages: 2 + Canconrtl weeds Disadvantage: Ensironmente A Iman uae of herbie Wh toety a more herbie eater we haematoma 2. Croegolnation wth wl plants onan crops > Resta new more resistant weed = “euparwande” 3 outcmpete and lone rational varieties of plants > toss of gona ders SoroHerbicide-Resistant Crops Gilyphosate-resstant crops a + Hah costo GM send 3 Too expensive for pple a buy farmers toe Genetic Techno > Might reduce efforts to reieve poverty GEL ELECTROPHORESIS Under developed counties become ore dependent on other counts Function: Te separate fragments of DNA according to length OR To separate dif proteins according to mass/charge Other ethical and social implications of (wea wo Gel Electrophoresis a ett a using GMOs in food production entation + posse are rescionn human / averse efecto the —- Immune sytem | Gel Electrophoresis — | CE | = tmtete Stauth eh t ne eve rer (OWA) Tole speci OMA egnenceGel Electrophoresis of DNA Procedure 1 Obtain suffer quant of NA + ONAcan amped wing PER or gene coningin 2.CutDNAInto fragment sng restriction eneymes ‘+ Many trapment a leraths produces 2. NA samples ace md with loading dye and Stalnng agent 2. Gels covered with butter solution Gel Electrophoresis of DNA sents ae loaded to wall the negative an of the go wth ONA opment frown lengths sul ran rg Gel Electrophoresis of DNA 6 DNAs tracted towards the post electrode (anode) + Seperation de to lc fil / potential derence + Bes DNA negatvaly charged ue tothe phosphate groups + Shorter agments of DNA move faster thar per oni ibe rough the lore toements + Duetolesimpedance of + NA rapments seperate and arnged Completed gel Gel Electrophoresis of DNA 2. Vein band under UV Bg snc Sting agent ates ONA to foesce tocrtimate length of agents Gel Electrophoresis of DNA Ce re ae er eae £ & 9. DNA heated to sepacgt strands IE = tT Torr LULL ut Gel Electrophoresis of DNA 10. DNA cooled nd gane probes ae ade 1 Gene probes are sed tolocate a pedi DKA sequence or game Gene probes = shor singe stranded DWA(ssONA) Labeled / tached wth radioactive /fuorescet substance + Cancomplamantary bate par with specie ONA fragments 21. Vew ONA fragments under UV gt / Xa fm (PAGE) instead of agarose + Sata rotaine migrate faster than ager poteneApplications of DNA Gel Electrophoresi - cesnemannannonne ull + nn faginng/ OA ring heme + ial ene + anti eeaning + breast ancer(BRCAL, BRAD) + Genes for haemphii, SCA, Huntington’ Diese and CF + Gone Therpy + eet uses, posomes naked ONA) + SCI, ented ee sas, CF Genetic Fingerprinting / DNA Profilin, E 1. stract ONA rom ts samples (4 Hood har oot, semen, saa) re Genetic Fingerprinting / DNA Profilin, f 2. deny up to 13 VNTR eons + Veriale Number f Tanda Rept (VNTR) thor ergs of repetitive noncoding econ of ONA + Number f epee ifr between indie + One rom each homelogus ehemosome 2. ant of A Increased by Polymerase Cha anc (PR) 4 0NA Fragmented by 5. Gal elacrophorsis of ONA-> ONA pecs trastared to membrane “> hosted to separate sacs > pane probe aed > Mile under U ight Xen fm > banding pattern sen Genetic Fingerprinting / DNA Profilin ( Eres berm oa sr = cae SoeGenetic Screening il 1 Preimplantation ent agnosis eee + Analysis person's DNA te check or presence fa particular allele + Ney formed emo rom in tv terisaton (vA) + Dua cttained rom sue samples. blood) + Tested before implantation int utes of mater + canbe cared out on embryo, fetus, newborn o ads + important examples: reas cance haemaphi, se cellanaemi, 1) Embryo bopey = removing rman embryo oungtor's seas, cst ies (CF), Down syne + masee sage (aay 4/5) 2) eR amply ONA iw! 4 2) 6atetacrophree 2 fn Ok tuyere ani set aes + Ee amon tings ne eo ‘) Preseacton» set olvembryo without aulty alles for implantation Genetic Screening 1. Pre-implantation genetic diagnosis vi =o won teen et | on om Seco etic impeons Steere Soatnpansten net eter 2 vty ahr antes om Mh eaecanton ace > Avo mplamavion of embryos wth ay ales [netomewnne —_|vbenby Allow couple tohave children whe woud ateraze hoor nto .carersoeenng condone "a secs Ethie concern: S)8)~) 6 + embryo might be datroyad nt prelates for mplanion 1+ oul ead to selection based on gender o spec wats (deste babes) contayto belt /sases, 2. Prenatal screening deg li ‘+ Helpsto provide warty dagnols or fetuses in taro > Socan ge ary weatment when ben In vitro fertilisation (IVF) 1. Hemee renner we lnc speowdon 2 Many corres tarase rom feat > Alls parents to prepare forthe bith of chi who wi ned vestmen for 3. Obtain tes / rose ser ‘onndarabetine or ven troughout le {© acy ed th span > Women can aoilate therapeutic abortions = terminate pregnancy for = Mealy genetic dierent sedealresane ‘ons Sooner e ae — nm se snl ees ane ted s surgi mtbr bed le» ~ OR eee embryos andstreforlngtimein frozen 20 [Iv + Frozen 20s s/f yb con/ea OOo3. Newborn screening / 4. Carrier Scree Eg. haemophia, sede cell anaemia cytes (CF, Huntngon’s disease, Down synsome a treasteancer é + Fuyalleles ofthe BRAY nd BRCA2 genes + Increate the hance of vlog breast cancer 1 created testing to detec cance ay + Bective mastectomy = removal of beasts) bore the ceurrnce/alapiousofeaneer |= 3. Newborn screening / 4. Carrier Scree imine lees * t ctccnteencoenerctonnanin | kt RES + Helps provide cay dagnsis ‘2g: Huntngton’s disease (yams oly occur tr in e) 1 ifpesent enables Meta change at tretment/ regular chk 2. prevertntve treatment my be cheer than esting ease eh “ested negate, gene sceening removes are 3. Newborn screening / 4. Carrier Scree Dieaeontges + Many mutations unknown somay Itperon ete post: + Mayland to aieey + May erperene soca anc Adu tat to model radi erry utr of protein DNA sequencing Bioinformatics 2 sepbextons uk 1 Venti evolutionary clatonsip/ goat relatedness sequence and protein haves ster inhumane +g Plumodiam, tophyococs rus + Canhelpinvaccoe/drug development