is Le Danie! Lim Pe A2 Biology 9700 ~ Chapter R - Gene Technology Peter Ting Introduction ; The sequence of nucleotide bases on a DNA molecule makes up the _genetic cade. Each triplet of three bases code for particular amino acid] Using this code, cell can produce any one ofthe many proteins that it requires. One of the many remarkable features of the genetic code is that it is universal; the three bases CAC, for ‘example, will code for the amino acid valine whether its in a bacterial cell ora cell from a human liver. A gene taken from one organism should then be able to produce ‘exactly the same productifitis introduced into another organism. This is the basis of ‘genetic engineering, the production of new characteristics by the insertion of a gene | Comment 1): coon ay | Isreal three A aes ton in the min ster ramacpion from one organism into another. D po BORE Ngee vile Genetic Engineering is widely used in today's society. Their purposes are; 2) defect & screen diseases Insulin romth hormones 3 gm shemry Vaccine este ross Insecticide resistance herbicide resistance Cate Therapy) 7 (Farming and Crop Production) vel non eee phylogeny ofextingt animal can be studied her Wansiopsatanee: by replicating DNA na bacteria ell Man nul ees Saeeiee lhaes improve quality of crops identi biological sample Detection and Sereening [angehans nthe panerens (Yo should |e te from AZ N= Repos oma a The wpe tf gl, £4. sping cole Key points: In 1921, dlabetic patients, whose elevated sugar eves are du to impaired insulin production, have been treated with insulin derived from the pancreas glands of “He Abe “cenvertin of exces ‘animals, eg. cow or pigs. The hormone, produced and secreted by the beta cells ofthe’ o cee glycyer- in Ver els pancreas’ lets of Langerhans, regulates the se and storage of food, particularly carbohydrates. Although bovine and porcine insulin are similar to human insulin, their "Sct ry gy hi composition is slightly different. Consequently, there were adverse effects. wi rake a ays rae Bovire insulin — Atom cans Tn diabetic prticnis, Hocus Mt heal in tine hema Freie insulin -ram: pic ‘hey ei ae | ve the yequiad substiates, Tete 'ncive thln Aram yigs may hove 4h be any wtertel-‘A2 Biology 9700 - Chapter R - Gene Technology Peter Ting ‘Animals who maybe lft prevcisty wil vansit ‘re pathogen together wiht nin “oo ile amount of inulin an be estate from animal | Take up to much space and cost and cos tor Due tania inst not being 00K ential wth tn anal est too much Raman ns, humans may develop antiboies aginst anima insulin Leong mical feasible These factors led researchers to consider synthesising insulin by inserting the insulin pe feneina a sata vector eg the cl bacterial ceo produce aninsin thats, away gone and Cotee iF Chemically identical tots naturally produced counterpart (bce) This has been eer aia Renaee TRAC euUEET THE Ree Gta CRE Oe OG ie, heping get fe sustainable method than extracting and purifying the animal product. bacteria prduce. the pretein ‘Steps of insulin production via recombinant DNA technology, Unaing i fia cell) oe ! Nake DMA Frey yn aoa rN Te ~ The DWA mate is cDth- ae ke Daring arscripiae, ints Q. =] 4 Veids in Dk a tema, x 3 DW contains Cons and shan? Only e¥4s are invohed a Hanscription eto 1 i i Spl mechan span partic Swnnotes Gene - Specific Sequence of mucleolile bares the cades for - ine paly peptide i : 2 Gere for insulin 6 feud in all sumatic. cell hocanse aif e Sinatic cele ovty The Some genetic infirmation. ‘ nen a 2 bettl , Ditferentiotion prodces different Spa of cells. They Apress Wh ae Ree : differant genes. Bit we obtain insulin gene fram @ coll bocowse it has active (PA and th Bel gee, so they actively ascribe Rwy, We Bae ENe ee Mie MRK, sytheid Teilin ane fam mANA. WE chise mRWk over BNA because ill be longer. Fiettn made it is te abedereniicania a h will a bee desied-one. Ck at SS‘A2 Biology 9700 - Chapter R - Gene Technology Peter Ting step 1L. identifying the human insulin gene oom 2. isolating mRNA and making CDNA using reverse transcriptase Comment [Mik wat, | ayers metic ae town a NA: 3, dloning the DNA using DNA polymerase ‘pigether wil | eetsaer ua payee ssDNA ce 1,2,3 Foes hanclice ieeeuiacuvicasae We nie, 99 pmee | moaned on rin Use Reverse Transcriptase to make rnnerese AY Sige hale IN inet area arava tent, gee ge eve Sm peaches Ge te - rN) Teale Th ba ees Use RRA REIDABDY produce cDNA complementary ‘ils ofp sarc Dna & 4 sequence. A a a ee HF ssperete hc othe pene of interes ccna aki je 13 S isthere another way of isolating a gene other than using reverse transcriptase? ‘Break the cell membrane using mechanical and chemical method (eg. vigorous shakir Cut DNA “thon gD oe Eco R1) > What's are me? > Note: Must cut outside of gene w/o too much “excess. baggage” 3. Separate DNA fragments by gel electrophoresis + Gel electrophoresis wil separate restriction fragments (digested DNA) into respective sizes. 4, Denature the DNA—? Wonetwe all dbyble shonded DN ‘= Treating the gel with alkali lice NaOH will break the dsDNA to ssDNA. Note — do. not use heating to denature the dsDNA while its stil on the agarose gel 5. Transfer DNA from the fragile gel to a nylon sheet 6. _Hybridize gene of interest with a radio-labeled DNA* or mRNA* probe and expose w/ film tolocate gene Se NeXt page > How do these probes work? (fig. 20.10) ‘6. Use PCR to clone the isolated gene of interest. Ownnotes A 6 As cut ohh mltale ® we Ke “ ogres % H ols DNA of T Suga hsphate boddhine St) polymerase eye on ‘Seoup a gemeciom when ake a ‘Comment [5] erm eyes Rerticin enzyes ie DMAsasiogensymes funda ectrs (an havece om er fore) Bese hey carvan te mle they re ten {std ste endoniesses Inordrtate eto sequence DNA Ris Ses ncosary ect int sna ftazments Hon) DNA digeteg names {kr thve nour panretic ui} ca {a buemactof then are mo ise | Decs produced bya gvenestion | [endentiane neon petite ‘tute aon hed a eaTaeREd dibs -Gath cc- Splicing mednicin : LE) aye CH Ke (toi) & cs hereaa F thy ae read in og Poy sos ae re vaurtig Slpueree 7p, GC x Mlndosic septence, | Je1ce ‘A2 Biology 9700 - Chapter R - Gene Technology a 2 (comment wt But eeemarese “ ‘ges \Oo arene oe 5 Dp: | a5 sectees P voters fnew efi: |" = ee SS Salem tebe Brel stuck ae | ad Balhae ine iohamic | eer ts ‘ie, 5 a Sete uct, Bs he sershisie ae Se puts gore GPG Matic chet je he 2 HM Hh Hh a —— sbi 1 |] —— " mE A a), OS Atsine cin Aer fal insect pees cha meee ewe 9) ase ot Sif pbce- ren oes ae Dn tetan—_enicn ems ee aA ree Mea ge —_| Sem man Tbe & odo hhelod - Yon step allius — Horessed- Siaccretomeony co WV IGA, ww : Sep ieee ARO ge Real a i Here df iclaesy‘A2 Biology 9700 ~ Chapter R - Gene Technology. Peter Ting, plasmid vector using fe eee © 1. Digest a plasmid vector with a restriction enzyme (e.g, EcoRI) ata single site to produce two sticky ends. 2, Digest human DNA with EcoRl to produce pleces with the same sticky ends ‘a. Use Human DNA or cDNA copied from mRNA using reverse transcriptase from retroviruses. 3. Mix the two samples and allow to hybridize ‘a. Some plasmids will hybridize with pieces of human DNA atthe EcoRI site. 4. Use DNA ligase is used to covalent link the fragments. Why plasmids are suitable vehicle for cloning? (tee cong vet + Plasmids are naturally occurring extrachromosomal DNA molecules. + Plasmids are circular, double-stranded DNA. + Plasmids are the means by which antibiotic resistance is often transferred from one bacteria to another. + Plasmids can be cleaved by restriction Newer cloning veto ‘enzymes, leaving sticky or blunt ends. + Artificial plasmids can be constructed by linking new DNA fragments to the sticky ‘ends of plasmid. ‘Own notes‘A2 Biology 9700 - Chapter R - Gene Technology + Acloning vector is a plasmid that can be modified to carry new genes. * Plasmids useful as cloning vectors must have: — An origin of replication. Should be capable of replicating in host cell = A selectable marker (antibiotic resistance gene, such as amp! and tet’) to indicate which host cells received recombinant ONA molecule. — Multiple cloning site (MCS) ‘where insertion of foreign DNA will not disrupt replication or inactivate essential markers + Should have convenient RE sites for inserting DNA of interest — Easy to purify away from host DNA. Fain mas) DNA cal Pid Vater = ¢€ (eae === 6 ©: [estan Peter Ting. DNA ligase seals the sugar- ~~ phosphate backbone Own notes{2 tog 9700- Caper Gene Tehlgy Peter ng sep 3. Inserting te plasmid ver inoteer -( )* ce Rees oa er ai 5 Inserting plasmid vector into host bacterium is known as transformation ‘Transformation isthe process of altering a cell so that it acquires and expresses genetic ‘material acquired from the surrounding environment. Transformation is commonty used to introduce recombinant plasmid DNA into bacterial strains which can transform naturally or can be made by competent for transformation by artificial means. These ee ee ee eee ‘There are 2 ways know to make cells to become competent. & Chemically Competent cells + Chemically competent cells Menbure are calcium chloride-treated Mi cd to facilitate attachment of feted coqe the plasmid DNA to the 4s offerct DIA competent cell membrane. ye uring chemical Which ' 194% -anstormation, the cells are heatshocked in a water bath; 1 which opens the pores of the (Co gin cell membrane allowing entry ., of plasmid DNA from the Upc bain but ICA nin Electrocompetent Cells + Electrocompetent cells are prepared for transformation using electroporation, a method that uses an electrical pulse to create Pores through which genetic material enters the cells. ‘Upon the completion of transforming the cells, there are afew valid questions that "must be answered before we proceed to expressing the protein out ofthe cells 1. Only some of the bacteria take up 2 plasmid—How do you know which ones did? 2. Notall plasmids are recombinant plasmids—How do you find those that are? 3. Only some of plasmids contain the gene of interest—How do you identify these? Own notes 7 cal - Cele fot can pick Me, He plaseid ype o calls con be prods cells take op plasnid Lut ‘Te yh Te@nbinant Alh us AY ope open ¥p s at if 7 es a tte 3. The celle rake yw plasmids nf Technane tm ot‘A2 Biology 9700 ~ Chapter R - Gene Technology Peter Ting step 6. identifying genetically Tositin has Ferting and modified bacteria using aN antibiotic resi Norte hoy amioeresnanc res Groctenory 2% polypeptile ha cnoncore ~ReGues Gigi boty 4 be symtbegeed ratoilly Vow +H spleck bacenia cel With vecarbinant DNA Ace anibinies sr eee ia select the recombinant cells you desire, you can accomplish that through the use of ‘antibiotic resistance genes found in the plasmid itself, Aer transformation, cells are plated onto agar medium that contains selective antibiotic: only transformed cells, that acquire antibiotic resistance gene on the vector plasmid, will survive and form colonies. All the untransformed cells will di. ie {he periplasm pace where breaks dw ‘een allowing el wal stesso Having two antibiotic resistance genes in tordomt eee the plasmid vector allows us to select ‘esntonce gene between recombinant cells wit plasmid aloneand recombinant cells with recombinant plasmid. Howis that possible? Referto the igure beside All you have to do s to place the gene of interest into tetracycline resistance gene region using restrietion enzyme and DNA ligase. ‘agin ‘stance gone fhe gene of interest is successfully cloned inside the plasmid vector, the recombinant cells should not grow on the plate containing both tetracycline and ampicilin, wae) ' Own notes oO Ce) ak ag ; ' Helniglin ks caliny O is bot ve Oss wt & becteia Aansfarned cl teaalnas‘A2 Biology 9700 ~ Chapter R ~ Gene Technology Peter Ting Step ". cloning the bacteria and harvesting the human insulin; “The gene expression of insulin inside a bacterial cell is not that straight forward, assuming that you have the gene of interest cloned inside the cel, This is because insulin has two polypeptide chains. *Q= Why is itnot “so straight forward"? (Own notes ic caey fakery ote cel has 1 Gayl appacactes =Figalln cane nately become canylefe in bectetcl tll i We synthesis the 2 chains | % etl ant then compre em 40 make insulin adie | b By sreatingin vite (ovtsile epuivérmeat) | Gimpice adive fosula (5 | syed |‘A2 Biology 9700 ~ Chapter R - Gene Technology __ eee “Key points: The process of recombinant DNA technology in making Insulin offers far ‘more advantages compared to extracting insulin from animals. Peter Ting baer ca epee by may fl root wea ces lee Inslin roduced wth bacteria work on human inelin gene, ‘therefore thers preduced by bacteria 10% ential wh human : i ED SS boctera canbe repeated easy and does .——_yy ee ‘Key potnts: Promoters are DNA sequences located in the adjacent region to the transcriptional start site. RNA polymerase and accessory proteins (transcription rods wi abe enpoend i cating (Suances toh anon becaute he fay i contd sly factors) bind to the promoter to initiate production of an mRNA transcript Interactione- of proteins at the promoter regulate gene activity by activating or repressing, transcription. ‘Own notes = We. « patente ri nwt From itees ae Te Foued sicecty —Binde ty dip ner nthiate dnuscay ee —Wihet gees dante CHGS aN’ Xhe cell wont hrake insslin 200 kilodaltons, as well as DNA fragments >100 basepairs = Note: Agarose gel allows separation of DNA across an electric field. The gels inert matrix acts as a sieve for DNA molecules, Smaller DNA usually will ‘migrate or move through this inert matrix faster than bigger DNA ‘A.comb is putin the gel to create holes which we call Sample was added with glyeerol/suerose, substance tomake DNA more dense to allow ito remain atthe bottom ofthe well Own notes Large ond soll PNA Fingers an be seperated by" gel Aectopaves's- B‘A2 Biology 9700 ~ Chapter R - Gene Technology Peter Ting ‘The sample will be loaded into ‘each well, and . Boorse. DINK is negatively charged- pide ane ae Beal curet aman ihe guding ur chamber Ay Kh aie Eas . > Blue dye (mixture of DNA fragments runs through the agar. Immediately after done running, the agar will be soaked with a chemical, ethidium bromide for staining purpe ‘Own notes attach IM Spat whe WV light & appl, Plasteapnce ts seh£A2 Biology 9700 - Chapter R - Gene Technology Peter Ting DNA fragments areluminated under UV light A computer system would be able to ‘capture and save the photo ofthe gel for further analysis {Besides showing DNA fragments, what other funeton can the electrophoresis. provider AON gerrining and DNA seueneig i bg chews an the a eleceaphresis We Rave DMA fim DNAfingerprinting— Pam pA $0. nee xk acnine—o hS 1 i ate Saeclk age evel (canvsnimal isthe prder of iden pcre Therd ars oo many ellis fees pairs in each person's DNA that every person has a different sequence: Itis called a “fingerprint” because itis very unlikely that any 2 people would have exactly the same DNA information, in the same way that itis very unlikely that any 2 people would have exactly the same physical fingerprint. The testis used to determine Whether a family relationship exists between two people, to identify organisms causing. a disease, and to solve crimes, Restriction fragment length polymorphism (RFLP) - This concept is about cutting a particular region of DNA with known variability (2 or more DNA strands which are NOT identical) , with restriction enzymes, then separating the DNA fragments by agarose gel electrophoresis and determining the number of fragments and relative sizes. The pattern of fragment sizes will differ for each individual (each DNA strand) tested, Own notes 45 Ti eye fing ake DNA fam Shard pein wll hove, different banding petteenRenved segs id ee OCT TT Tie 25 Fon Tht ee ee eae ea ane cel thee & he pre alfa “8 meas wre. ‘ be A2 Biology 9700 ~ Chapter R - Gene Technology Peter Ting ‘The 2 samples was treated with ONE = restriction enzyme. ‘The first sample has a recognition site ‘sequence for the enzyme but the second doesn't Therefore producing <== varying number of fragments and sizes. This is called restriction fragment length polymorphism ‘hiss how paternity testing is done too Using this concept, a target DNA sequence is selected This target sequence is unique and that everyone must heve it. The concept of inheritance s thatthe biological child must inherit the chromosomes fromthe (y+ isp ofthe parent, one each from each pair. Lstlaqs (pao = euh wer Sid ke) # ® \ayls et bak cease Comme 1 Terme ews ‘ penne aca Ads 05 « “ley Somewtape rains ‘Shwe pe 1)Probe tact the trae 2 Stal eae 2 Llabeoes a 3 Sell 1 me ht ah cen rapid pin ve emia FEL biological child of Jack and jill? Explain why? Swe nates —For each advidual, the r8trictien enegne, cols at JG Event plicy on Di ~ St thee © wique tunbor A teams anf Sue ok each frag mort for each jadi vidal TE Is ner possible, fo AEnttky Aue individ with stoe gaelic dkeep ¥ rer dwins have dibfertt guetic malkeyp so cam be tetified 16‘AZ Biology 9700 - Chapter R - Gene Technology Peter Ting DNA sequencing First thing you need to know ishow ‘ANTP works. ddNTP stands for dideoxymucleotide. t's where the normal DNA molecule that you already learned in Genetic Control (AS); lacks another “O" from their hydroxyl group at carbon 3 of ‘heir sugar. ‘What isthe significance? If they lack "OH" ‘group, (Which is the basis of forming a phosphodiester bond with another phosphate group from another nucleotide), a ddNTP cannot form bonds with another nucleotide, AT ALL. Therefore the any growing polynucleotide chain that has a ddNTP attached to it, will stop growing Te dideoty & mel s « Growing chain, He dais Sis riving, ot Not firm bands ue 1 5h if ancthor wait aoe oF an EXAMPLE? Ske (GTR Te Ge -KKL XXX RK = ! eae 4 Cary it OWA ropli atan Behe UA ao gear rT ee ee ‘Next a primer is annealed to one of the template strands. This primer is specifically ‘constructed so that itis located next to the DNA sequence of interest. Once the primer Scatached to the DNA, the sluonsdvde into fur tubeslabelgd 6% 1" a at | tong aed te tase spel Rin on Ad6 ICT GBTtP, AdcTT bind. ‘tube: all four GNTP's, ddGTP and DNA polymerase "A" tube: al four ¢NTP's, ddATP and DNA polymerase "T tube: al four GNTP's, dATTP and DNA polymerase °C’ tube: all four dNTP's, pump Sa ates Keypoints Cystic Mbrosi (CF) sa genetic condition resuting froma mutation ina a igene that codes fora transporter protein called Cystic Fibrosis Transmembrane it Regulator (CFTR). This protein lies Inthe cell surface membrane of eels in many parts of the body, including the lungs, pancreas and reproductive organs. The CFTR eter ee when elt can atl Ors Namally chloride oasae transparted ft of the cli through the PTR prot Water follows by osmosis. When the CFTR protein is not working, this daes not Arey can caate| Salt Poenerd, Seppe There thereon nate onthe errs ftecctanteresbosld <4 gag alee wend be The mucus ls produced in these areas therefore does not mx with water inthe en rst be Lept hyatal to Plea teeie oe eee : = Gort ajc le \ Game ee iit ms a y, Vis cbfetens & smah.S can Sweep Them anny Alsi gh Bie hohe pie see) g ion Whe 6 Ligyte gene found in chromosome 7 Yelelion o& 3 base pois ©‘A2 Biology 9700 ~ Chapter R ~ Gene Technology aoe eal gene des nahin men, Fg Mots Seabee os Bh, 0 nm ai — Trsed ne, S peda Ro, eee LY ao ie | ay os ae an5 DNA encapsulated in lipid micelle Generally liposomes are smal. so may Interfere withthe number of DNA it can carry Prvied yeducs Ae Get gene Genial) with th suctheg Vigostne or 7 6nd relete ins “ef od ere nbn longer regenerating Strictly speaking, genetic testing is used with individuals who, perhaps because of their family background, believe that they, their children or future children es ‘may be a risk of carrying the gene for a genetic disease. Genetic screening involves wider scale testing of populations to “cpio pose individuals that are at risk ofa genetic disease. 7 There & «snail Ge of mé calage- Digadvardayes = Gere trasttrayel thn Nom befere. Sah les 2 a aan SwtyNe Cyipseic art _ ba inject Mem my anne ace tn ‘They ae specfc and eficet in delivering the gee ito target call" POH - stra care must be taken to ensure that Wal partes are n0 Peter Ting wal veckis ave mig hk gre jc Bective becawe wus 7 “anne genetic mateal 4s jer He col ith the cel ruckus. But the — produe nove Vitas ‘fect 4h 20« ‘A2 Biology 9700 - Chapter R - Gene Technology Peter Ting [Feeimique | Whatitinvolves Abnormality detected [Amniocentesis [Removal ofsmall | Down's Syndrome volume of amniotic Mid] Spina bifida that bathes the fetus and contains cells from the fetus. The proteins in the fui can be analyzed as they may be abnormal in spina bifida fo the number of chromosomes can be| counted (Down's syndrome sufferers have 3 copies of Huntington's disease| Muscular dystrophy proteins [Maternal serum |Tests mother's blood | Spina bifida screening | for presence of fetal |Utrasonographs| Techniques for gaining | Organ shnormalitics landfetoscopy” | an image ofthe fess ‘Advantages + If negative, reduces uncertainty and fear ‘and may increase lfe-expectancy + allows sorting of 1VF embryos or PGD + individuals ean identify if they are carriers ofa harmful gene before they have children + allows informed decision about IVF /eag/embryo donation so fewer children ‘with genetic disease will be born + lower long-term health costs + frequency of harmful alleles reduced + allows early diagnosis of disease + allows earller treatment of the disease allows parents to prepare ‘emotionally/financially for affected children ‘Own notes roantye Disadvantages some test procedures involve risk + presence of abnormality may not affect health * positive result causes stress and may lead to emotional problems of both affected individual and family members + risk of false negative false positive + may lead to an increase in abortions + against religious beliefs + costly to administer ‘an be used by insurers/employers to iminate against people a