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CNG NGH SINH HC I CNG
CHNG 1: KHI NIM C BN V CNG NGH SINH HC

1.1. Khi nim v cng ngh sinh hc C nhiu nh ngha v cng ngh sinh hc (Biotechnology), ty theo tng tc gi khc nhau, nhng tt c u thng nht v khi nim c bn sau y: Cng ngh sinh hc l s sn xut cc sn phm trn quy m cng nghip, trong nhn t tham gia trc tip v quyt nh l cc t bo sng (vi sinh vt, thc vt, ng vt). Mi t bo sng ca c th sinh vt hot ng trong lnh vc sn xut ny c xem nh mt l phn ng nh. Vo nhng nm 1980, cng ngh sinh hc chuyn sang mt giai on mi l l giai on cng ngh sinh hc hin i vi vic s dng cc thnh tu ca k thut gen, l lnh vc cng nghip s dng hot ng sinh hc ca cc t bo c bin i di truyn. C s sinh hc c p dng y bao gm sinh hc phn t, sinh hc t bo, ha sinh hc, di truyn hc, vi sinh vt hc, min dch hc, cng cc nguyn l k thut my tnh... C hai cch nh ngha cng ngh sinh hc mt cch tng qut nht: - Do UNESCO (1985) nh ngha: Cng ngh sinh hc l cng ngh s dng mt b phn hay t bo ring r ca c th sinh vt vo vic khai thc sn phm ca chng. - Do Trng Lut Stanford (1995) nh ngha: Cng ngh sinh hc l cng ngh chuyn mt hay nhiu gen vo sinh vt ch nhm mc ch khai thc sn phm v chc nng ca gen . S khc bit r rt nht ca hai nh ngha trn thuc v i tng tc ng ca cng ngh sinh hc: UNESCO xem c quan, b phn, t bo v chc nng ring r ca sinh vt l i tng, trong khi Trng Lut Stanford li coi gen l i tng tc ng ca cng ngh. T cc nh ngha trn, c th phn bit c hai nhm cng ngh sinh hc l: - Cng ngh sinh hc truyn thng (Traditional Biotechnology) Bao gm: + Thc phm ln men truyn thng (Food of Traditional Fermentations) + Cng ngh ln men vi sinh vt (Microbial Fermentation Technology) + Sn xut phn bn v thuc tr su vi sinh vt (Production of Microbial Fertilizer and Pesticide) + Sn xut sinh khi giu protein (Protein-rich Biomass Production) + Nhn ging v tnh bng nui cy m v t bo thc vt (Plant Micropropagation) + Th tinh nhn to (In vitro Fertilization) - Cng ngh sinh hc hin i (Modern Biotechnology) Bao gm:

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+ Nghin cu genome (Genomics) + Nghin cu proteome (Proteomics) + Thc vt v ng vt chuyn gen (Transgenic Animal and Plant) + ng vt nhn bn (Animal Cloning) + Chip gen (DNA chip) + Liu php t bo v gen (Gen and Cell Therapy) + Cng ngh sinh hc nano (Nanobiotechnology) + Tin sinh hc (Bioinformatics) + Hot cht sinh hc (Bioactive Compounds) + Protein bit dc (Therapeutic Protein) S phn loi cng ngh sinh hc cng c th da vo cc tc nhn sinh hc tham gia vo qu trnh cng ngh, c th chia thnh cc nhm sau: - Cng ngh sinh hc thc vt (Plant Biotechnology) - Cng ngh sinh hc ng vt (Animal Biotechnology) - Cng ngh sinh hc vi sinh vt (Microbial Biotechnology) - Cng ngh sinh hc enzyme hay cng ngh enzyme (Enzyme Biotechnology) Gn y, i vi cc tc nhn sinh hc di t bo cn hnh thnh khi nim cng ngh protein (Protein Engineering) v cng ngh gen (Gen Engineering). Cng ngh Protein v cng ngh gen xuyn sut v tr thnh cng ngh cha kha nm trong cng ngh sinh hc thc vt, cng ngh sinh hc ng vt v cng ngh sinh hc vi sinh vt. Nh k thut c trnh t gen v k thut DNA ti t hp, cng ngh gen t c nhng thnh tu ht sc to ln mang tnh quyt nh, m ra nhng giai on pht trin mi. l nghin cu v ton b genome ca nhiu sinh vt, trong ng ch l vic gii m genome ca con ngi v ca cy la. l vic hnh thnh c mt phng hng nghin cu, ng dng v kinh doanh cc sinh vt chuyn gen (Gentically Modified Organism-GMO) v cc thc phm chuyn gen (Gentically Modified Food-GMF). Cng ngh protein c tim nng ng dng rt ln trong vic sn xut ra cc protein ti t hp (Recombinant Protein) dng lm dc phm iu tr cc bnh him ngho nh: interferon, interleukin, insulin... Mt khc, ty vo i tng phc v ca cng ngh sinh hc, c th phn ra cc lnh vc cng ngh sinh hc khc nhau nh: - Cng ngh sinh hc nng nghip (Biotechnology in Agriculture) - Cng ngh sinh hc ch bin thc phm (Biotecnology in Food Processing) - Cng ngh sinh hc y dc (Biotechnology in Medicine-Pharmaceutics) - Cng ngh sinh hc mi trng (Environmental Biotechnology) - Cng ngh sinh hc vt liu (Material Biotechnology) - Cng ngh sinh hc ha hc (Biotechnology in Chemical Production) - Cng ngh sinh hc nng lng (Biotechnology in Energy Production)...

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1.2. Lnh vc nghin cu ca cng ngh sinh hc Bn lnh vc cng ngh sinh hc hin nay ang c quan tm nhiu nht l:. 1. Cng ngh sinh hc trong nng nghip L lnh vc cng ngh sinh hc c nhiu ng gp trong vic ci thin ging cy trng, xy dng nhng k thut canh tc mi, nghin cu qu trnh c nh m nhng cy khng thuc h u... - Cng ngh sinh hc trong ci thin v nhn nhanh ging cy trng. Lnh vc ny c bn ng dng chnh: ng dng k thut chn dng t bo bin d soma, nhn ging trong ng nghim (nhn ging in vitro), lai v tnh hay cn gi l dung hp t bo trn, k thut sn xut cy n bi (1n). - C nh m v bin np gen nif. Dng k thut gen tch gen nif t cc c th c nh m chuyn sang cc cy trng quan trng nh la, ng l mt m hnh l tng ca cc nh to ging. - Cc phng php canh tc mi, bao gm: Phng php mng dinh dng, h thng thy canh. - Cng ngh sinh hc trong chn nui, bao gm: K thut cy chuyn phi, to ch phm phng trnh bnh cho ng vt... 2. Cng ngh sinh hc trong y dc Nhiu cng trnh nghin cu ca cng ngh sinh hc c ng dng thnh cng trong y dc, c bit l trong sn xut thuc v trong chun on bnh. Trong nhng nm qua, lnh vc ng dng cng ngh di truyn mnh nht trong y t l nghnh sn xut thuc khng sinh, vc xin, khng th n dng v cc protein c hot tnh sinh hc. Hin nay, cc nghin cu nhm tm kim cc cht khng sinh mi tng mnh do hin tng vi sinh vt khng li tc dng ca khng sinh ngy cng nhiu hn. Phm vi ng dng ca khng th n dng trong ngnh y t ngy cng tng nh phn tch min dch, nh v cc khi u, pht hin mt s protein c lin quan n s hnh thnh khi u, xc nh s c mt ca cc loi vi khun khc nhau, ... gip cho cc bc s xc nh bnh mt cch nhanh chng v chnh xc. Khng th n dng l tp hp cc phn t khng th ng nht v mt cu trc v tnh cht. Khng th n dng c to ra bng cch cho lai t bo lympho trong h min dch ca ng vt hoc ca ngi vi t bo ung th. Mt s th lai c kh nng to ra khng th c hiu i vi khng nguyn. Chn cc th lai nhn ln v sn xut khng th n dng. Cc t bo lai c kh nng tng sinh vnh vin trong mi trng nui cy - tnh cht ny nhn c t t bo ung th. Nh cng ngh s dng DNA ti t hp m ngi ta c th sn xut mt s protein c hot tnh sinh hc dng cha bnh nh insulin cha bnh tiu ng, interferon cha bnh ung th, cc hormon tng trng cho con ngi. Bn cht ca cng ngh ny l lm thay i b my di truyn ca t bo bng cch a gen m ha cho mt protein c hiu v bt n hot ng to ra mt lng ln loi protein m con ngi cn.

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3. Cng ngh sinh hc trong ch bin thc phm Cng ngh ln men l mt lnh vc quan trng trong sn xut thc phm. Vic tuyn chn cc chng vi sinh vt c kh nng ln men tt, em li hiu qu cao l rt cn thit. Cc nghin cu s dng cng ngh di truyn phc v cho cng ngh ln men ch yu i vo hai hng chnh l: - Phn tch di truyn cc loi vi sinh vt s dng trong qu trnh ln men, xc nh cc gen m ha cho cc tnh trng mong mun nhm to ra nng sut v cht lng sn phm ln men. - To ra cc vi sinh vt chuyn gen phc v cho cc qui trnh ln men. V d trong sn xut ru, ngy nay ngi ta dng cc chng vi sinh vt c kh nng to ru cao v cho hng v tt. Phn ln cc chng c nghin cu, tuyn chn, lai to bng cng ngh di truyn. sn xut ru vang, trc y, ngi ta phi dng hai loi vi sinh vt l S. Cerevisiae to ra hm lng ru trong dch ln men v sau , s dng Leuconostos trong ln men ph qu trnh tng tr, nhm nng cao cht lng ca ru. Ngy nay, ngi ta tin ti dng mt chng vi sinh vt chuyn gen thc hin c hai qu trnh. i vi cc sn phm ln men sa nh phomat v sa chua, trc kia, ngi ta thng s dng nhng vi sinh vt t nhin c mt trong sa ln men, do vy, ngi ta kh lng kim sot qu trnh ln men v hiu qu khng cao. Ngy nay ngi ta to c cc chng mi vi cc tnh cht xc nh v iu khin c qu trnh ln men theo nh hng mong mun. Bng cng ngh vi sinh vt, cng ngh gen ngi ta to ra nhng chng vi sinh vt c kh nng tng hp cc enzyme chu nhit, chu axit, chu kim tt sn xut enzyme. Enzyme -amylase chu nhit v ang c s dng nhiu sn xut nha, ng glucose t tinh bt. Trc y, trong cng nghip thc phm cc nghin cu cng ngh sinh hc c s dng ch yu hon thin cc quy trnh cng ngh ln men truyn thng. Cn hin nay, cc nghin cu cng ngh sinh hc ch yu lin quan n vic to ra cc chng mi c nng sut sinh hc cao v vic p dng chng vo cc cng ngh ln men hin i, trong sn xut v ch bin cc loi sn phm sau: - Cng nghip sn xut sa. - Cng ngh sinh hc trong ch bin tinh bt. - Sn xut nc ung ln men, nh: bia, ru nho, ru chng ct... - Sn phm cha protein, nh: protein vi khun n bo, protein t to lam c nh m cyanobacteria v vi to. - Sn xut cc cht tng hng v thc phm, nh: citric acid, amino acid, vitamin v mu thc phm, cht tng v ngt thc phm, keo thc phm... - Ch bin rau qu. 4. Cng ngh sinh hc bo v mi trng Cng vi s pht trin ca khoa hc k thut, loi ngi phi bt u tm cch gii quyt vn nhim mi trng bng cc bin php khc nhau. Trong , cc bin php cng ngh sinh hc ngy cng t ra u vit hn so vi cc bin

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php khc. Ni chung, hin nay vn bo v mi trng c gii quyt theo ba hng sau: - Phn hy cc c cht v c v hu c. - Phc hi cc chu trnh trao i cht ca C, N, P v S trong t nhin. - Thu nhn cc sn phm c gi tr dng nhin liu hoc cc hp cht hu c. - X l cht thi, nh: x l sinh hc hiu kh, x l bng ln men phn hy ym kh. - Thu nhn cc cht c ch t ln men ym kh, nh: x l cc dng nc thi khc nhau v ti s dng chng phc v cho cc ngnh cng nghip nng. - X l cc cht thi cng nghip, nh: x l cht thi cng nghip ch bin sa, x l cht thi cng nghip dt. 1.3. Lc s pht trin ca cng ngh sinh hc Lch s hnh thnh v pht trin cng ngh sinh hc tri qua cc giai on sau: 1. Giai on th nht hnh thnh t rt lu trong vic s dng cc phng php ln men vi sinh vt ch bin v bo qun thc phm, v d: sn xut pho mt, dm n, lm bnh m, nc chm, sn xut ru bia Trong , ngh nu bia c vai tr rt ng k. Ngay t cui th k 19, Pasteur ch ra rng vi sinh vt ng vai tr quyt nh trong qu trnh ln men. Kt qu nghin cu ca Pasteur l c s cho s pht trin ca ngnh cng nghip ln men sn xut dung mi hu c nh aceton, ethanol, butanol, isopropanol vo cui th k 19, u th k 20. 2. Giai on th hai Ni bt nht ca qu trnh pht trin cng ngh sinh hc trong giai on ny l s hnh thnh nn cng nghip sn xut thuc khng sinh penicillin, khi u gn lin vi tn tui ca Fleming, Florey v Chain (1940). Trong thi k ny xut hin mt s ci tin v mt k thut v thit b ln men v trng cho php tng ng k hiu sut ln men. Cc th nghim x l cht thi bng bn hot tnh v cng ngh ln men ym kh to biogas cha ch yu kh methane, CO2 v to ngun phn bn hu c c gi tr cng c tin hnh v hon thin. 3. Giai on th ba Bt u t nhng nm 50 ca th k 20, song song vi vic hon thin cc quy trnh cng ngh sinh hc truyn thng c t trc, mt s hng nghin cu v pht trin cng ngh sinh hc hnh thnh v pht trin mnh m nh mt lot nhng pht minh quan trng trong ngnh sinh hc ni chung v sinh hc phn t ni ring. l vic ln u tin xc nh c cu trc ca protein (insulin), xy dng m hnh cu trc xon kp ca phn t DNA (1953). Tip theo l vic tng hp thnh cng protein (1963-1965) v c bit l vic tng hp thnh cng gen v buc n th hin trong t bo vi sinh vt (1980). Chnh nhng pht minh ny to tin cho s pht trin nhanh chng ca cc nghin cu c bn v ng dng thc t sau trong lnh vc cng ngh sinh hc hin i.

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4. Giai on t t K t 1973, khi nhng th nghim khi u dn n s ra i ca k thut DNA ti t hp c thc hin v s xut hin insulin-sn phm u tin ca n vo nm 1982, v th nghim chuyn gen vo cy trng nm 1982 thnh cng th n nay cng ngh sinh hc hin i c nhng bc tin khng l trong cc lnh vc nng nghip (ci thin ging cy trng...), y dc (liu php gen, liu php protein, chn on bnh...), cng nghip thc phm (ci thin cc chng vi sinh vt...)... Cng ngh sinh hc pht trin cho n ngy nay ch yu da trn ba cng ngh chnh l: - Cng ngh vi sinh. - Cng ngh t bo (nui cy m v t bo...). - Cng ngh sinh hc hin i, tc cng ngh gen. Cng c tc gi gn qu trnh pht trin cng ngh sinh hc vi ba cuc cch mng sinh hc. - Cch mng sinh hc ln th nht (u th k 20): s dng qu trnh ln men sn xut cc sn phm nh acetone, glycerine, citric acid, riboflavin... - Cch mng sinh hc ln th hai (sau th chin th 2): sn xut khng sinh, cc sn phm ln men cng nghip nh glutamic acid, cc polysaccharide, trong c thnh tu v t bin, to cc chng vi sinh vt cho nng sut v hiu qu cao, pht trin cc qu trnh ln men lin tc v pht hin phng php mi v bt ng enzyme s dng nhiu ln... - Cch mng sinh hc ln th ba (bt u t gia thp nin 1970): vi cc pht hin quan trng v enzyme hn ch, enzyme gn, s dng plasmid lm vector to dng, t nn mng cho mt nn cng ngh sinh hc hon ton mi l cng ngh DNA ti t hp. Hai giai on u, cng ngh vi sinh v cng ngh t bo, s dng hot ng sinh hc ca cc t bo tch bit, nhng cha bin i c cu trc di truyn ca chng, nn c xem l hai giai on ca cng ngh sinh hc truyn thng. Phi n cuc cch mng sinh hc ln th ba nh nu trn, th mi ra i nn cng ngh sinh hc hin i, giai on pht trin cao nht ca cng ngh sinh hc, m ra k nguyn mi ca sinh hc. 1.4. V tr ca cng ngh gen trong cng ngh sinh hc Cng ngh gen l mt mi nhn ca cng ngh sinh hc. Nm 1972-1973 k thut gen ra i, m u cho mt giai on mi ca cuc cch mng cng ngh sinh hc. Con ngi c kh nng vt gii hn tin ho, c th chuyn cc gen t mt loi ny sang mt loi khc, iu m khng th thc hin c bng con ng chn lc t nhin. n nay ngi ta gii m b gen ca mhiu sinh vt nh b gen ca vi khun E. coli, ca nhiu virus, ca giun trn, ca cy la v n nm 2003 v c bn gii m c b gen ngi. Trn c s k thut gen cc nh khoa hc to ra nhiu ging cy trng c nhng c tnh u vit nh cy chng chu hn, chu mn, cy c nng sut cao

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gp phn l tng gi tr kinh t cho x hi. Nhiu chng vi sinh vt mi c kh nng tng hp enzym cao c to ra bng cng ngh gen c s dng trong cng nghip. S hiu bit mt cch chi tit v cu to v vn hnh ca b gen cho php con ngi c kh nng can thip mt cch chnh xc vo cc qu trnh sng phc v li ch ca mnh. Nhng thnh tu ca cng ngh gen lun gn vi ng dng thc tin nhm phc v li ch ca con ngi. Tm li c th ni cng ngh gen l mi nhn, l trung tm pht trin ca cng ngh sinh hc. 1.5. Khi nim v t bo gc Thng 2 nm 1997, Wilmut cng b cng trnh nhn bn v tnh ng vt, to ra c con cu Dolly, nh mt du mc cho s pht trin v nhn ging nhiu loi ng vt t t bo dinh dng (t bo soma). Nm 1999 t bo gc (Somatic stem cell) c phat hin. T bo gc l nhng t bo vn gi nguyn tnh cht nh t bo phi, tc l chng c kh nng pht trin thnh nhiu loi t bo chuyn bit khc nhau nh t bo tim, t bo thn kinh, t bo thn vv... v t c th pht trin thnh mt c th. Nh vy nhn bn v tnh ng vt t t bo gc s d dng hn nhiu. T n nay vic nhn bn v tnh ng vt t cc t bo gc c nhiu nh khoa hc trn th gii quan tm. Nhng thnh cng trong nhn bn v tnh ng vt m ra kh nng nhn bn v tnh con ngi. y l mt vn c nhiu nh khoa hc, nh qun l, nhng ngi ng u ca quc gia quan tm bn ci v cho n nay c mt s nc cm khng cho tin hnh nhn bn ngi. 1.6. Thc phm chuyn gen Cho n nay nhiu loi sinh vt chuyn gen (GMO) xut hin vi qui m sn xut cng nghip, nh cy ng chuyn gen, cy u tng chuyn gen, c chua, la vv...Nhng cy chuyn gen thng c nhiu c tnh u vit nh nng sut cao, c th chng chu nhng iu kin ngoi cnh bt li nh chng chu su bnh, chng chu hn, chu mn vv...Nh vy khi trng cy chuyn gen thng em li hiu qu kinh t cao. Cy trng l thc phm cho ngi v ng vt nn phi chu s gim st ca cc t chc qun l v an ton sinh hc, v vy nhng loi cy chuyn gen a ra sn xut phi m bo nhng tiu chun nht nh. V d M ni m cng ngh sinh hc c u t v pht trin mnh nht trn th gii, vn qun l cng rt cht ch H thng qun l ca M nhm m bo an ton lng thc bao gm B Nng nghip M (USDA), Cc Bo v Mi trng (EPA), Cc qun l Thc phm v Dc phm (FDA), qun l cc loi lng thc c ngun gc thc vt c to ra nh cng ngh sinh hc. Theo o lut Lng thc, Dc phm v M phm (FD&C), FDA c thm quyn bo m an ton ca tt c cc lng thc trong nc v nhp khu cho ngi v ng vt trn th trng M. C quan Kim tra Sc kho Thc vt v ng vt ca USDA (APHIS) c chc nng gim

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st an ton nng nghip v an ton mi trng trong trng trt v th nghim ti hin trng cc ging cy trng c to ra nh cng ngh sinh hc. Tuy vy cng cn lu rng vic to ra cc GMO, cc gen khng khng sinh nh kanamycine, ampicillin hoc hygromycine thng c dng km lm gen ch th. Chng tn ti trong sn phm ca cc GMO v c th c nh hng trc tip hoc gin tip thng qua dy chuyn thc n ca sinh quyn n con ngi. 1.7. Mt s kha cnh v kinh t v khoa hc ca cng ngh sinh hc hin i Cc phng tin thng tin i chng ng ti khng t cc kin phn i ng dng mt s thnh tu cng ngh sinh hc trong sn xut, thm ch i vi nhng thnh tu c gii khoa hc nh gi l sng chi. Tht vy, cng ngh sinh hc cng nh khoa hc ht nhn, bn cnh cc ng dng cc k to ln cho li ch v pht trin ca loi ngi, c th cn mang li nhiu him ha khng th lng trc c hu qu. Sau y chng ta s tm hiu cc him ha tim tng ca cng ngh sinh hc. 1. V khoa hc S d dt trong s dng cc sn phm chuyn gen lm thc phm cho ngi v gia sc c th do nhiu l do khc nhau, nhng tu trung c th chia thnh hai nhm sau: - B my di truyn ca sinh vt mang tnh hon thin rt cao v tin ha qua hng trm triu nm, nhng gen mi c lp thm vo cho cy trng v vt nui tng nng sut hoc cht lng nng sn, bit u c th ph v tnh hon thin, tnh cn bng ca s sng cc sinh vt ny. V v th con ngi khng th yn tm vi vic hng ngy nut vo c th mt s lng ln cc sn phm thiu tgnh hon thin, cn bng hay ni cch khc l c th c d tt. - Cho n nay trong vic to ra cc GMO, cc gen khng khng sinh nh kanamycine, ampicillin hoc hygromycine thng c dng km lm gen ch th. Chng tn ti trong sn phm ca cc GMO v c th c nh hng trc tip hoc gin tip thng qua dy chuyn thc n ca sinh quyn n con ngi. Hin nay, ngi ta ang tm cch thay th cc gen chn lc c bng cc gen c v t hi hn nh gen m ho protein pht hunh quang mu xanh lc (Green Fluorescence Protein-GFP). Gen GFP c coi l mt gen ch th tt, v n lm cho cc GMO pht sng xanh rc r khi t di tia t ngoi. Nhng d sao s nghi ngi vn cn, v gen GFP c ngun gc t mt loi c Bc Bng Dng, ch khng t mt ng vt c ngun gc gn vi ngi. 2. V kinh t 2.1. Nhng cng ty a quc gia v cng ngh sinh hc T chc quc t nng nghip tin b RAFI (Rural Advancement Foundation International) l mt t chc quc t phi chnh ph Canada hot ng nhm hn ch nh hng ca cc cng ty a quc gia v ging. Theo RAFI, th k 21 s l nhng nm tung honh ngang dc ca cc cng ty a quc gia v cng ngh sinh hc, hin nay nhng cng ty ny ang pht trin

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nhanh chng nh thu tm cc cng ty nh hn v trc ht nh li nhun khng l thu c trong c quyn bn cc sn phm GMO. Chng hn cch y hn 15 nm, cng ty Monsanto ch chuyn v cc sn phm ha du, thuc tr su, tr c. Tuy nhin thi gian gn y, Monsanto u t rt ln v trin khai cng ngh gen thc vt to ra cc ging GMO v ang tr thnh cng ty ging ln nht th gii. RAFI gi Monsanto l mt "Microsoft cng ngh sinh hc" v t nm 1996 n nay Monsanto mua li nhiu cng ty trc y vn l ngi khng l trn th trng ht ging. 2.2. S l thuc vo cc cng ty a quc gia v cng ngh sinh hc RAFI tin on ngi nng dn hu ht cc nc trn th gii, k c cc nc cng nghip pht trin, s dn dn s b l thuc vo mt nhm nh cc cng ty cng ngh sinh hc a quc gia. Vi quy ch ngt ngho v quyn tc gi IPR (Intellectual Property Right) hin hnh trong quan h kinh t th gii, ngi nng dn s b tc b hon ton quyn t do trng cy g trn mnh t ca mnh v bn cho ai sn phm ca mnh. L do cc cng ty nh Monsanto c c nhiu quyn hn nh vy chnh l s tin b ca cng ngh sinh hc. Chng hn, gen terminator c c quan ng k bn quyn ca M chnh thc cp bng pht minh cho cng ty Delta Pine (3/1998). Khi chuyn gen vo bt c mt ging cy no, ht bn ra s ch ny mm trong mt th h duy nht. Nu ngi nng dn ly ht trng v sau, gen ny s to ra mt hp cht git cht mm, v th ht hon ton khng ny mm c. Vi gen terminator trong tay, cc cng ty a quc gia s bt nng dn cc nc hng nm phi mua ht ging ca h. Mt khc, cc cng ty ging ang thn tnh dn cc cng ty ch bin lng thc, thc phm l u ra ca sn phm nng nghip. Mt mt c quyn ht ging GMO, mt mt nm cc cng ty ch bin nng sn, cc cng ty a quc gia cng ngh sinh hc s khng cha mt li thot no cho nng dn cc nc ang pht trin. 1.8. Nhng vn php l ca cng ngh sinh hc hin i K thut ti t hp DNA gip cc nh khoa hc thay i c ch tin ha ca t nhin, sng to ra sn phm ca gen, to ra cc dng sinh vt mi. Ngy cng c nhiu bng chng hin nhin v li ch ca k thut DNA ti t hp. Nhng cng phi cn nhc n nhng nguy c tim tng ca n, v thc t cng ny sinh mt s vn php l quan trng buc chng ta phi xem xt li mt cch thn trng. C nhiu vn php l trong cng ngh sinh hc, tuy nhin trong phm vi bi ging ny ch cp n mt s kha cnh sau: 1. Vn an ton sinh hc Mc ch ca cng ngh sinh hc l phc v cho li ch ca con ngi. Tuy vy nhiu vn ny sinh ra khi tc ng n cc sinh vt, mt s khng t ca chng l nhng k th ca con ngi nh cc vi sinh vt gy bnh. Vn an ton sinh hc v o l c s quan tm ca x hi. Cng ngh gen em li nhiu

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li ch cho con ngi, tuy nhin bn cnh cng khng t nhng ni lo ngi, do vy vn an ton sinh hc c t ra ngay t nhng ngy u. An ton sinh hc l s bo v con ngi, x hi v mi trng khi tc ng c hi, tc ng nguy him do cc c t hay cc sn phm ca cng ngh gen gy ra cho hm nay v cc th h mai sau. N i hi phi nh gi mc an ton ca tt c cc sn phm cng nh cc bin php s dng i vi vt nui cy trng, cc liu php cha tr i vi con ngi. Ngay t nhng nm u ca cng ngh gen (1972-1973) mi lo ngi v s xut hin mt loi vi sinh vt mi nguy him cho con ngi v ng vt c th xy ra lm cho cc nh nghin cu dng cc th nghim ca mnh. Ngay t vn an ton sinh hc c t ra. Ngy nay cc nh nghin cu phi tun th theo cc nguyn tc ch o qun l chnh thc c a ra nhm m bo cc vi sinh vt ti t hp ang nghin cu khng pht trin bn ngoi cc phng th nghim, ng thi cc nhn vin phng th nghim phi c bo v khng xy ra bt c mt s ri ro no. Cc sn phm chuyn gen lun c kim tra, th nghim mt cch nghim ngt trc khi a vo s dng. V d nm 1999, c khong 6000 th nghim ngoi thin nhin c thc hin ti M v mt s c chp nhn. Tuy nhin cc nh mi trng lun lo lng v cng c nghin cu qu quyt rng khi cho chut n khoai ty chuyn gen th h thng min dch ca n b tn thng, hay ht phn t cy bp chuyn gen Bt hu hoi qun th bm "hong hu", hay bi kch ca bnh vim no b Anh v mt s nc ....Kt qu ca nhng thng tin ny dy ln nhng phn ng chng li nhng cy chuyn gen. 2. Qun l v nh gi an ton ca thc phm Ti M v mt s nc pht trin vn qun l v nh gi an ton ca thc phm rt nghim ngt. Tnh an ton ca cc lng thc t cy trng cng nh thnh phn thc phm, bao gm hng v, cht ph gia, thnh phn dinh dng, cc cht gy d ng vv... phi c nh gi k trc khi cho php tiu th. Tuy vy i khi cng c nhng l hng trong qun l, cng nh li nhun cao lm gim i phn no tnh nghim ngt, do vy nhng vn tranh ci lun tn ti nh vic s dng tryptophan gy ra hi chng au c b nghi ng l c sn xut t dng vi khun to ra bng cng ngh gen hay vic s dng hormon tng trng b (Somatotropin b vit tc l BST). Ngi ta thy rng khi tim BST cho b sa th s lm tng ng k lng sa v lng BST tn d trong sa khng cao so vi i chng nn khng c hi cho con ngi, tuy nhin BST t nhin thu nhn vi gi thnh cao nn BST sn xut nng cng ngh gen ra i (rBST). Cc nh qun l v nh khoa hc M kim tra rt k v c kt lun l tht v sa b c x l vi rBST an ton cho ngi. Tuy nhin nhng ngi phn i s dung rBST cho rng vic s dng rBST s lm gia tng nhim vi khun vo tuyn sa gy chng vim v. Tranh ci su hn na nh vy khi s dung rBST th phi dng khng sinh cao hn n nh sc kho ca gia sc dn n lm tng d lng khng sinh trong tht v sa, iu ny c th gy d ng nhiu ngi tiu

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dng. Do vy mc d FDA ca M khng nh l an ton nhng nhiu quc gia nh Canada v cc nc chu u t chi s dng. 3. Quyn tc gi Nhng cuc tranh lun din n quc t v quyn tc gi ca cc nc c ngun gen qu him c phng Ty s dng trong cng ngh to ging, khng em li mt kt qu no. 169 nc ng k vo cng c Quc t v a dng sinh hc (Convention on Biological Diversity) v cng c ny c hiu lc t 12/1993, trong quy nh cng chia s quyn li gia cc nc c ngun gen vi cc cng ty phng Ty s dng ngun gen . Tuy nhin, t n nay cc nc c ngun gen qu him vn tip tc b mt dn ti sn quc gia ca mnh m quyn li c chia s th khng ng k. Chng hn, ngy 16/1/1996, Bn quyn s hu s 5.484.889 ca M c cp cho Gio s Sinh hc phn t (i hc New York) Sylvia Lee-Huang bo v quyn tc gi ca gio s v mt loi protein chit t cy Momordica charantia. Cy ny l mt loi ti nguyn c hu min Nam Trung Quc, c ngi Trung Quc gi l da ng, l thnh phn chnh ca mt bi thuc dn gian chng nhim trng c lch s hng th k. Lee-Huang tuyn b: "Nh cng ngh DNA ti t hp, t nay khng bao gi chng ti cn mua ht da ng t Trung Quc, v cc protein ti t hp sn xut trong phng th nghim i hc New York hon ton ging nh protein chit t qu da ng trc y". Nm 1994, hng ArgEvo phn lp c gen PAT (phosphinothricin acetyltransferase) t dng vi khun Streptomyces viridochromogens c trong mu t ly t Camerun. Gen PAT cho php to ra cc ging cy trng khng thuc dit c nhm glufosinate, ng gp quan trng vo doanh s 2,3 t USD ca AgrEvo nm 1995. Tuy nhin, hng ny t chi khng tr cho Camerun mt khon tin no v quyn tc gi.

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CHNG 2: CNG NGH DNA TI T HP

2.1- Khi nim DNA ti t hp 2.1.1- Khi nim 1- DNA: DNA l ch vit tt ca axit 2-deoxyribonucleic, c nh sinh hc ngi Thy in Miescher tm ra vo nm 1869 trong nhn t bo bch cu (t bo m). u tin ng gi n l nuclein c ngha l hch nhn. Sau , ng pht hin ra n c bn cht axit v gi n l axit nucleic. Sau hn 80 nm cng vi s tranh ci ca nhiu nh khoa hc, n nm 1952, ngi ta mi cng nhn DNA l vt cht di truyn. DNA gm 3 thnh phn chnh: baz nit dng purine v pyrimidine (A,T,C,G), ng pentose (ng 5 carbon) v axit phosphoric (H3PO4). Ba thnh phn ny c t l 1:1:1 v chng lin kt vi nhau to thnh mt nucleotide:
Oh Ho P ch2 5 H H 3 O O H A(t,c,g) H

Hnh 2-1: Cu to mt nucleotide

Oh

Cu trc ca DNA c Watson-Crick khm ph ra. l chui xon kp cong nh nhng, c cu trc khng gian 3 chiu, gm hai si song song, i xng v b sung cho nhau theo mt qui lut nghim ngt: A bt cp vi T v C bt cp vi G nh cc lin kt hydro. Cu trc xon kp ca Watson- Crick l chic cha kha m ra nhng k thut ca s sng. 2,- DNA ti t hp: Vi cu trc c bit ca phn t DNA v s bt cp nghim ngt ca cc baz nit, nhiu nh nghin cu ny ra tng: nu to ra c nhng ui mt trong hai si ca phn t DNA khc nhau th chng c th ni li vi nhau nh bt cp b sung. Vo nhng nm 70, bng nhng phng php khc nhau, ngi ta to c nhng phn t DNA ti t hp u tin. Nm 1972, nhm nghin cu ca trng i hc Stranford (Paul-Berg) a ra phng php ui ni cc phn t DNA khc nhau. to u dnh, ngi ta s dng enzyme terminal transferase. Di tc dng ca enzyme ny, ngi ta to c nhng ui poly nucleotide khong 50 n 100 gc adenine

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(polyA) u OH (3) ca mch n DNA SV 40 (Simian virus 40) v ui polyT DNA plasmid c m bi enzyme. Sau khi trn ln hai loi DNA ny, cc ui b sung adinine v thymine bt cp li vi nhau v vi s c mt ca enzyme ligase, hai DNA ny c ni li v to ra plasmid ti t hp vng. V s an ton cho cng ng nn sn phm ti t hp ny khng c bin np trong t bo ch. Tuy cha hon tt, nhng thc nghim ca Berg cho ta hai vn mu cht v DNA ti t hp. ng cho rng, c th dng enzyme ct DNA mt cch nh trc v cc on DNA cc loi khc nhau c th ni li vi nhau. Nm 1973, Staley Cohen v Annie Chang to ra nhng phn t DNA ti t hp t cc loi khc nhau, c nhiu u im v c bin np trong t bo ch. V t y, cng ngh DNA ti t hp ra i. Sau , nhiu nh khoa hc lao vo cc th nghim lp ghp gen v nhanh chng thu c nhng kt qu c ng dng trong thc tin. K thut ti t hp DNA do Rerg, Chang, Boyer v Cohen ra gip cc nh sinh hc c th lm thay i tnh di truyn ca c th sng theo chiu hng nh trc v vt qua c hng ro ngn cn loi. Mi t hp DNA mi u to nn nhng c im sinh hc mi k c v mt di truyn ln sinh ha. Vy ngi ta gi DNA ti t hp l mt DNA lai, tm c invitro (trong ng nghim) bng cch t hp 2 DNA thuc 2 loi khc nhau. V d: Hai vector l plasmid, phage c ci mnh DNA l to ra DNA ti t hp (Hnh 6-2). Cc DNA l ny c gi l on ci hay DNA ngoi lai. N chnh l mt mnh (on) DNA hay l gen m ngi nghin cu quan tm v ghp n vo DNA ca plasmid hay DNA ca virus.

DNA plasmid DNA l DNA phage

Vector

Plasmid Phage DNA l

Hnh 2-2: M hnh biu din s to ra DNA ti t hp 2.1.2- Mc ch to DNA ti t hp DNA ti t hp c ch tc vi mc ch l thc hin mt qu trnh gi l s tch dng. Tch dng l s tch lp v thu nhn nhiu bn sao ng nht ca mt gen hay ca mt mnh on DNA. Tch dng c th c i km hoc khng c i km vi s biu hin protein. Nhng p dng ny ca DNA ti t hp

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dng thit lp cc ngn hng b gen, cDNA hoc tng hp protein, enzyme, hormone, ... 2.1.3- Nhng yu cu ca DNA ti t hp - DNA ti t hp phi t c nhng yu cu cn thit ca mt vector chuyn gen. - DNA ti t hp phi c hot tnh khi a vo t bo ch. - DNA ti t hp phi c nhng du hiu d pht hin trong qun th vi khun. V d quan st s hot ng v biu hin ca gen ti t hp. 2.2- Cc cng on chnh to DNA ti t hp 2.2.1- Chn nguyn liu DNA c chn lm phng tin vn chuyn gen (vector) thng l DNA plasmid v DNA phage. Chng c thu nhn ch yu t t bo vi sinh vt, c tch v lm sch theo phng php chit tch DNA plasmid, DNA virus. DNA cha gen cn nghin cu (DNA l) cng c thu nhn t t bo sinh vt c chit tch v lm sch v kim tra theo phng php chit tch DNA. Ngoi ra, ngi ta cn s dng DNA tng hp bng phng php ha hc hoc tng hp t mRNA nh enzyme sao chp ngc theo c ch nuclease S1 hoc k thut Gubler-Hoffman. Cc enzyme ct, ni cc on DNA li vi nhau nh enzyme ristriction, enzyme ligase cng vi s hp tc ca mt s enzyme khc nh kinase v alkanline phosphotase, ... 2.2.2 Cng on ct vi s tham gia ca enzyme RE kiu II RE (Restriction Endonuclease) kiu II: l nhng enzyme ct hn ch nhng v tr xc nh v c dng trong cng ngh DNA ti t hp bi v chng c mt s u im: Hu nh ch c mt kiu hot tnh ct, enzyme khc m nhim vic ci bin. Mi mt enzyme ct v tr ph hp nh trc ngay bn trong hay cnh trnh t nhn bit. Chng ch cn ion Mg+2 v khng cn nng lng ATP. V d: EcoRI 5'G A A T T C 3' 3'C T T A A G 5' - Cc enzyme ny gp chui ch ca n trn phn t DNA v ct DNA thnh hai mnh dng u bng hay u dnh. Hiu qu ct ca enzyme gii hn ph thuc vo nhiu yu t khc nhau nh hm lng ca enzyme, nhit , ion kim loi, mi trng m, pH v tinh khit ca DNA. 2.2.3- Cng on ni vi s c mt ca DNA ligase Bc tip theo ca k thut di truyn l ni cc on DNA vo vector chuyn gen to plasmid c mang DNA l. Phn ng ni c thc hin nh enzyme DNA ligase. DNA ligase l mt enzyme xc tc cc phn ng ni hai mnh DNA bng cch to cu ni phosphodiester gia u 5(P) v u 3(OH) ca hai nucleotide

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ng cnh nhau (Hnh 6-2). Trong sinh hc phn t, ngi ta coi DNA ligase nh mt cht keo phn t kt dnh cc mu DNA li vi nhau. 1,- Ni u bng: Ni u bng c xy ra khi hai on DNA u bng ng cnh nhau. Di tc dng ca enzyme ligase, lin kt phosphodiester gia u 5(P) v u 3(OH) c hnh thnh v hai nucleotide c ni li vi nhau (Hnh 6-3a). Kh nng ni hai on DNA u bng rt thp. tng hiu sut phn ng, thng ngi ta phi tng nng ca cc on DNA. 2,- Ni u lch: u tin, hai mnh DNA u lch do c nhng baz b sung nn chng tin gn li nhau to lin kt hydro gia cc baz nit b sung. Sau , hai nucleotide ca hai u ni to lin kt este gia OH(3) v P(5) di tc dng ca enzyme DNA ligase. Phn ng ni c thc hin theo s c biu din trn Hnh 2-3b. Plasmid c tch t t bo E. Coli hoc t bo nm men theo phng php chit tch DNA plasmid. Sau , dng enzyme RE II (nh EcoRI) ct to ra plasmid h c hai u lch nhau. 2.3 Cc phng php to AND ti t hp 2.3.1. Phng php n gin dng u lch 1,- Chn v x l AND plasmid Plasmid c tch t t bo E. coli hoc t bo nm men theo phng php chit tch AND plasmid. Sau dng enzym RE II ct to ra plasmid h c hai u lch 2,- Chn v x l on ci: DNA on ci c thu nhn v la chn, tinh ch theo mc ch ca tng nghin cu. Ct DNA bng enzyme RE loi II cng loi vi enzyme ct plasmid (EcoRI) to ra cc vt ct ging nhau. 3,- To plasmid ti t hp: Ghp on ci vo plasmid bng cch cc DNA on ci vi plasmid vi s c mt ca enzyme DNA ligase, ta thu c DNA ti t hp. Phn ng ni xy ra theo s c biu din trn Hnh 2-4.

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Ni u bng:
OH P 5

C C G G

+
5 P 3 OH

A T T A
OH P

C C G G

A T T A
P OH

DNA ligase
C C A T G G T A

Ni u lch:
G C C T A G G A T C T A

Bt cp baz b sung

P OH 3 5

G G A T C T C C T A G A

DNA ligase

G A T C T A
5 OH 3 P

C C T A G

Hnh 2-3: DNA v cc phn ng ni

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2.3.2- Cc phng php s dng on ni 1,- Chn v x l vector plasmid: (tng t nh trn) 2,- Chn v x l on ci: Linker l nhng on DNA di 10 n 15 nucleotide, c tng hp bng phng php ha hc, sao cho gia mi linker phi c trnh t nhn bit bi mt enzyme ct hn ch loi II. V d nh EcoRI c chui ch l G/AATTC. cDNA u bng c tng hp t mRNA nh enzyme sao chp ngc theo c ch nuclease S1. Metyl ha cc vng hn ch c trong on cDNA si i c nhn bit bi enzyme EcoRI. linker c cha chui ch G/AATTC vi cDNA v enzyme DNA ligase to phn t lai. Ct phn t DNA lai bi enzyme EcoRI, ta c phn t lai c hai u lch.

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DNA l

Ct bi Eco RI (REH ) Aatt Ct bi Eco RI (REH ) TtAa TtAa Aatt

Aa

tt

Hnh 2-4: Phng php dng u lch

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a Tt a
Tt aa

Aatt TtAa

DNA ligase

Tt

DnA ti t hp

Aa tt
aa

aa Tt

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im nhn bit ca Eco RI C C G A A T T C GG GG C T T A A G C C

Linker

Cc phn t linker

DNA l (cDNA)

DNA ligase

DNA plasmid

Eco RI

Eco RI

DnA ti t hp

Hnh 2-5: Dng cc on ni linker to DNA ti t hp

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3,- To vector ti t hp: to vector ti t hp, ngi ta ghp phn t DNA lai u lch vo plasmid m cng c ct bi cng mt enzyme EcoRI. Nh tc dng ca enzyme DNA ligase m vector ti t hp c to thnh (Hnh 6-5). 2.3.3- Phng php s dng enzyme terminal transferase (ETT) Nguyn tc: Da vo c tnh ca enzyme terminal transferase c kh nng gn cng mt loi nucleotide vo u 3(OH) ca phn t DNA. Cch to DNA ti t hp bng phng php ny c tin hnh theo 4 bc sau (Hnh 6-6): 1,- Bc 1: Chn v phn lp DNA l u bng v plasmid, gi s ta phn lp plasmid c cha chui ch c nhn bit bi enzyme BamHI (G/GATCC). Ct plasmid bng enzyme BamHI to plasmid h c hai u dnh. Ct DNA l bng enzyme exonuclease theo hng 5 3 to DNA c hai u lch nhau. 2,- Bc 2: DNA l vi cng mt loi nucleotide dCTP vi s c mt ca enzyme terminal transferase to ui polyC u 3(OH) ca DNA l. plasmid vi cng mt loi nucleotide dGTP vi s c mt ca enzyme terminal transferase to ui polyG u 3(OH) ca plasmid h. 3,- Bc 3: a DNA l vo plasmid vi s c mt ca enzyme DNA ligase, cc u mt ca homopolymer c trnh t b sung (---GGGG 3/3CCCC---) s bt cp vi nhau. 4,- Bc 4: B sung enzyme DNA-polymerase I gn cc nucleotide tng ng vo ch trng theo nguyn tc b sung. Enzyme ligase ni lin kt phosphodiester v cui cng to c plasmid ti t hp c hai chui ch c nhn bit bi enzyme BamHI. u im ca phng php l ghp DNA l u bng vo plasmid to DNA ti t hp v ch tc c DNA u lch t DNA u bng.

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3 5 5 3 Ct DNA bng E exonuclease 5 3 3 5 PolyC ETT CCC CCC 5 3 GGA T C C C C T A GG Plasmid cha chui ch ca BamHI

Ct bng E BamHI (3) GA T C C G G C C T A G (3)

PolyG ETT (3) GGGGGA T C C G C C T A GGGGG (3) G

CCC G C C T A GGGGG

GGGGGA T C C CCC G

BamHI GGA T C C C C C C C T A G G GGG

BamHI GGGG G A T C C C C C C C T A GG DNA ti t hp

Linker

Linker

DNA l

Hnh 2-6: Cch to DNA ti t hp dng enzyme terminal transferase

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2.4- Mt s phng php a DNA ti t hp vo t bo ch 2.4.1- Ha bin np Hin tng bin np l cha kha gip ta hiu bit c s phn t ca gen, cng l cng c thc hin cc thao tc to tnh di truyn ca vt sng. Theo Mandel v Higa cho thy rng, E. Coli tr nn rt d b bin np bi DNA ngoi lai khi cc t bo vi khun c x l trong mi trng c CaCl2 v trc c sc nhit 42C. Ha bin np l phng php s dng cht ha hc, to iu kin a vector ti t hp vo t bo ch. Qu trnh c thc hin theo hai buc sau: X l t bo ch trong dung dch CaCl2 nhit thp nhm thay i mng t bo v vector ti t hp vi t bo ch x l. Hiu sut ca phng php ha bin np ny vo khong 105 n 106 t bo bin np trn 1mg DNA ti t hp. Qua cc kt qu thc nghim, ngi ta thy rng, cc t bo pht trin pha sm n pha gia d c bin np hn. Nhng nghin cu sau ny cho thy vic x l t bo bng cc ion kim loi ha tr hai nh Mg+2, Mn+2 v Ba+2 cng cho kh nng bin np ln. Ngoi ra, hiu sut bin np cn ph thuc vo kch thc ca plasmid, plasmid cng nh th hiu sut bin np cng cao. 2.4.2- in bin np Nguyn tc: S dng dng in cao th cc b theo xung to l nh trn mng sinh hc ca t bo, to iu kin cho t bo hp th DNA ti t hp c d dng. Hiu sut: T 109 n 1010 t bo bin np cho 1mg DNA ti t hp, tuy nhin, lng t bo bin np b cht nhiu c khi ln ti 70%. Hiu sut ca phng php ny ph thuc vo cc yu t sau: - mnh ca in trng tc ng khc nhau i vi cc loi t bo khc nhau, - di ca hng s thi gian (thi gian ngt xung - ms). Theo nhiu nghin cu, hu ht i vi cc loi t bo sinh vt, hiu qu bin np cao khi hng s thi gian t c khong 6ms, - Nng t bo ch, - Nng DNA ti t hp, - Giai on pht trin ca t bo (t bo qu gi hoc qu non u khng thch hp), - Mi trng dung dch m, - Cu trc mng t bo. 2.4.3- Bin np t bo trn (p rotoplast) L phng php chuyn DNA ti t hp vo t bo ch c x l bng polyetylen glycol (PEG). to t bo trn, ngi ta t bo vi PEG nng 30% n 40%. y l phng php chuyn gen c hiu qu cao i vi t bo thc vt. Bng phng php ny, ngi ta nhn c cc cy mang gen bin np n nh v di truyn qua nhiu th h (Potrykus v cng s - 1995).

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u im: Tn s bin np ng thi gen ch th v gen cn bin np cao. C th chuyn gen vo t bo protoplast ca bt k loi cy no. c bit l loi cy c gi tr kinh t cao nh la, ng, i mch. Nhc im: Vic ti sinh cy protoplast cn rt kh khn mt s loi cy. 2.4.4- Phng php bn gen Nguyn tc: Ngi ta s dng ht kim loi nng c bao bc DNA v bn trc tip vo t bo. u im: Phng php ny c th bin np cho tt c cc loi t bo thc vt. Thao tc d dng, bn mt ln c nhiu t bo. Nhc im: Hiu sut bin np thp, thng xuyn nhn c cy bin np khm (cy c t bo bin np v t bo khng bin np). Mt s thnh tu t c bng phng php bn gen: Nm 1988, Mc. Cabe v cng s nhn c cy u tng bin np u tin bng phng php bn gen. Nm 1990, Promm, Gordon, Kamm v cng s nhn c cy ng bin np nhiu phng th nghim. Nhng nm gn y c hng lot cng b v bin np thnh cng la. Nm 1996, Zthang v cng s bin np u , ma v bng. iu khng nh tnh u vit ca phng php ny. 2.4.5- Phng php vi tim Phng php vi tim l phng php s dng vi kim v knh hin vi a DNA ti t hp vo mi t bo nht nh. y l qu trnh lai ghp cho t bo bc cao hoc t bo hp t. Ty thuc tng trng hp c th, ngi ta c th chn phng php sao cho vic a DNA ti t hp vo t bo c hiu qu cao. u im: C th ti u lng DNA ti t hp a vo t bo v quyt nh a DNA vo loi t bo no. a chnh xc v thm ch vo tn nhn ca t bo v c th quan st c. Cc t bo c cu trc nh nh ht phn, t bo tin phi cng c th tin hnh mt cch chnh xc. C th bin np cho mi ging cy. Nhc im: Mt pht tim ch c mt t bo v thao tc cn phi kho lo v t m. 2.4.6- Ti np Ti np l hin tng chuyn vt liu di truyn qua vector l virus t vi khun cho sang vi khun nhn, trong c qu trnh chuyn gen v ti t hp gen vi khun nh thc khun th (Bacteriophage). Thc nghim chng minh c ti np E. Coli qua phage , phage P1 v Bacillus subtilis qua phage SP10 . So vi cc phng php trn, ti np cho hiu sut cao hn. Nh vy, n nay c nhiu phng php ha hc, ha l, c hc v sinh hc a DNA ti t hp vo t bo ch. Ty cc i tng v yu cu c th, phng php ny hay phng php khc c hiu qu v c s dng nhiu hn. 2.5- K thut tch dng (to dng)

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K thut tch dng bao gm vic phi ci mt mnh (chui) DNA l vo mt vector (plasmide hoc phage ) bng phng php ha sinh. Sau , a phn t lai ny vo t bo ch chn la bng phng php bin np hoc ti np. Trng hp mun to dng tng hp enzyme th mnh DNA nh ci phi m ha cho gen cu trc ca mt enzyme no . 2.5.1- Mc ch ca s tch dng - Thit lp ngn hng b gen, - Thit lp ngn hng cDNA, - Sn xut protein, enzyme, - Sn xut vaccine, - Sn xut khng sinh. 2.5.2- Cc bc c bn ca phng php tch dng Qu trnh thc hin c th thay i ph thuc vo nhn t tham gia v mc ch ca qu trnh tch dng. Tuy nhin to dng, cn phi thc hin cc bc sau: 2.5.2.1- Tch lp cc DNA l cn to dng Chn v ct DNA l ca t bo cho bng mt enzyme ct hn ch (RE). Phn lp on DNA (gen qu) ph hp vi vector v mc ch cn to dng. Trong trng hp c bit, to dng mt gen cha bit, ngi nghin cu c th tng hp ha hc on DNA cn to dng khi d on cu trc protein do gen ch huy tng hp hoc tng hp cDNA t mRNA. To u dnh khi cn thit. 2.5.2.2- Chn v x l vector Chn vector cn phi ch nhng yu cu sau: ln ca gen l (on ci), loi t bo ch tip nhn vector v phng php ng dng. X l vector: ct vector bng enzyme ct hn ch cng loi vi enzyme ct DNA ni trn ( to nhng vt ct ging nhau, thun tin cho vic ni ghp sau ny). Kh nhm phosphat bng enzyme alkanline phosphotase trnh hai u vector ng kn tr li. 2.5.2.3- To DNA ti t hp (Vector ti t hp) Vic to DNA ti t hp bng cch ghp DNA l vo vector c ct cng mt enzyme ct hn ch loi II, khi , chng s ghp i nhng u dnh li vi nhau nh bt cp b sung. Mt phn ng ghp ni xy ra vi s c mt ca enzyme DNA ligase ca E. Coli hoc phage T4 hon chnh phn t lai. 2.5.2.4- Chuyn DNA ti t hp vo t bo ch bng phng php bin np hoc ti np Cng on ny nhm mc ch s dng b my ca t bo ch sao chp vector ti t hp thnh mt s lng ln bn sao. Vic chuyn DNA ti t hp vo t bo vi khun tc l lm cho vi khun tr thnh kh bin, ngha l c kh nng thm vector ti t hp. S thm ny c th xy ra mt cch t nhin hoc c cm ng. Tuy nhin, n s ph thuc vo loi plasmid s dng lm vector v ph

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thuc vo s nh v ca vng ci lp cha bn trong vector m ngi nghin cu s chn phng php bin np hoc ti np. Bin np l hin tng chuyn vt cht di truyn trc tip t t bo th cho D (Doner) sang t bo th nhn R (Reception), khng cn s tip xc gia hai t bo hoc nhn t trung gian l phage hoc virus. Bin np c thc hin vi vector chuyn gen l plasmid. C nhiu phng php bin np nh ha bin np, in bin np, bin np t bo trn, phng php bn gen v phng php vi tim. Ti np l hin tng chuyn vt cht di truyn trc tip t t bo th cho D (Doner) sang t bo th nhn R (Reception) qua nhn t trung gian l virus. Ti np c thc hin vi vector chuyn gen c ngun gc l virus nh phage, cosmid, ... 2.5.2.5- Pht hin dng cn tm Cng vic tip theo l kim tra s hin din ca gen mong mun. Vic chn la nhng chng nh mun l khng n gin, v cch tin hnh th nghim trong mt hn hp khng ng nht nn cc dng vi khun c th mc ln theo ba kh nng: - T bo vi khun khng nhn plasmid ti t hp, - T bo nhn plasmid nhng khng c gen l, - T bo vi khun nhn c plasmid ti t hp. Ty thuc vo mc ch nghin cu m ngi ta c cc phng php khc nhau xc nh dng cn tm. Nu mc ch l nghin cu on gen cha bit, ngi ta thng dng u d. Nu on DNA bit (cDNA), cng vic s n gin v nhanh chng. 2.5.2.6- Kim tra v thu nhn sn phm ca gen ti t hp Ty tng trng hp c th m ngi nghin cu a ra nhng phng php kim tra thu hi sn phm ca gen ti t hp. Nu l mc ch thit lp ngn hng cDNA, ta phi tin hnh cc bc sau: u tin ngi ta phi tch plasmid ti t hp ra khi dch nui cy, sau , ct plasmid ti t hp bng enzyme RE loi II v thu c hai bng DNA, mt c di bng khung vector, cn bng khc c di tng ng ng bng cDNA. Cch kim tra li cDNA c ct bng cch in di trn gel agaroza 0,8% v kim tra li di ca nhng bng cDNA thu c. Nhng on c kch thc khc nhau s di chuyn nhng khong cch khc nhau trn gel. Nu sn phm ca gen ti t hp l protein, enzyme, hormone, vaccine, khng sinh,... ngi ta c th thu nhn sn phm bng phng php chit tch lng lc ly tm kt ta phn on hoc trao i ion. 2.6- Mt s phng php xc nh dng cn tm 2.6.1- Phng php lai axit nucleic 1,- Khi nim u d: Cc u d l nhng mnh DNA c nh du, c kh nng nhn bit mt chui DNA hoc RNA ng ng qua lai ha. 2,- c im ca u d:

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L mt on axit nucleic (DNA hoc RNA) si n. u d phi b sung cc baz nit, i song song vi on axit nucleic cn nhn bit. u d phi so mc c, l cc u nh du phng x. 3,- Cc loi u d: cDNA lm u d cc tt, ngoi ra, mRNA v oligonucleotide cng c th lm u d. Nu chui DNA cha bit trnh t, ngi ta phi tinh sch mt lng nh protein tng ng vi DNA nghin cu v t tng hp u d. Cn nu mt phn DNA phi so mc bit th cng vic rt d dng tng hp mt u d. 4,- Nguyn tc ca phng php lai axit nucleic: Da vo kh nng bin tnh khi nhit tng v hi tnh khi h nhit t t ca DNA. Khi tng nhit ca DNA ln qu nhit sinh l (thng khong 80 n 95C), hai si n DNA s tch ri nhau do lin kt hydro b t. Khi h nhit t t ca khi DNA bin tnh cng vi cc iu kin thch hp khc, cc mch n bt cp tr li. S bt cp ch c th xy ra khi hai trnh t DNA hon ton b sung cho nhau. Tnh c hiu cc ln ca phn ng lai ny cho php bt k trnh t mch n no cng tm gp mch b sung vi n, mc d chng nm trong hng triu cc trnh t DNA v RNA khc nhau. C th dng ng v phng x nh du pht hin on lai. 5,- Cch tin hnh: Chun b mu (u) d c nh du bng ng v phng x hoc ha cht. Bin tnh dng cn tm di dng mt si, sau h nhit t t, cc si n tng ng bt cp vi nhau. S bt cp xy ra gia DNA v DNA, RNA v DNA, RNA v RNA. 2.6.2- Phng php s dng khng th 1,- Nguyn tc: Da vo phn ng c trng ca mt s protein v khng th m chng ta c th nhn bit c qua phn ng c trng nh phn ng to mu, phn ng kt ta. 2,- Cch tin hnh: Chn khng th c trng cho protein (thuc th cho protein). Tch protein c biu hin trong qu trnh to dng. protein vi khng th c trng. Sau tin hnh nhn bit thng qua cc du hiu ca phn ng c trng gia protein v khng th. 3,- ng dng: Ch yu s dng trong y hc vi hai yu cu sau: DNA cn nghin cu c gn vo vector phi biu hin protein khi to dng v ng thi, vector to dng phi l vector biu hin (v d nh vector gt11). Protein biu hin cn phi c khng th c trng. 2.6.3- Phng php pht hin do mt hot tnh v xen on Nguyn tc: Da vo s mt kh nng khng thuc ca vi khun mang vector ti t hp khi chng c nui cy trong mi trng khng sinh.

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Phm vi s dng: Phng php ny dng cho vector c t hai gen khng thuc tr ln, v d nh vector pBR322 . Trong plasmid pBR322 c hai gen khng thuc AmPR v TetR. Trn cc gen ny c nhng im nhn bit ca cc RE ni ci t DNA l. Khi gn DNA l vo mt trong hai gen ni trn th gen nhn on ci mt kh nng khng thuc. Quan st nhng khun lc b mt gen khng thuc, chng t nhng khun lc c cha gen cn to dng. u im ca phng php l t tn km so vi phng php lai. 2.6.4- Phng php pht hin do thay i kiu hnh 1,- Nguyn tc: Da vo s thay i mu sc ca khun lc, chng t rng, khun lc c cha gen l. 2,- Cch tin hnh: Chun b hai mu th nghim m plasmid to dng c cha gen lacZ (v d nh dy pUC), mt mu so snh cn mu kia ci DNA l vo gen lacZ. Sau , nui cy c hai mu trong mi trng c cht cm ng X-Gal (5 brom4chloro-3indolyl D-galactopynoside). Cht cm ng ny b phn hy bi enzyme -galactosidase to ra galactose v X (dn xut indol), ngoi mi trng, dn xut ny b oxy ha cho mu xanh m. Qua quan st, ta thy plasmid khng cha gen l cho khun lc mu xanh do gen lacZ tng hp enzyme -galactosidase. Cn plasmid th 2 cho khun lc mu trng, iu chng t n cha DNA l c ci trong gen lacZ v chn nhng khun lc mu trng. 2.7- Ngn hng gen (Genomic library) Ngn hng b gen ca mt sinh vt l tp hp cc trnh t DNA cu thnh b gen c gn vo vector, n ch biu din tng s ca gen. Ngn hng ny c to ra t mt b su tp DNA ti t hp c cc chui m ha ln cc chui khng m ha. Khc vi ngn hng cDNA, ngn hng b gen c th c thit lp bt k loi t bo no ca sinh vt nghin cu. Cc bc thit lp ngn hng gen: - Tch v lm sch DNA ca sinh vt, - Ct DNA thnh nhng on c kch thc xc nh bng enzyme RE loi II thng thng, ngi ta s dng EcoRI, - Vector c chn to dng gen thng l cosmid hoc phage cng c x l bi EcoRI, - To vector ti t hp v ng gi trong v phage, - a vector ti t hp vo t bo ch v to dng, - Xc nh c tnh v biu hin ca dng vi khun va lp, - Tin hnh sng lc, ngha l phi tch nhng vector no c on DNA a chung, t xy dng ngn hng b gen. c im v ng dng: - Ngn hng gen cha cc on DNA ln hn 100 ln so vi cDNA. - Gii m thng tin di truyn cha trong b gen, c bit l cu trc intron v exon ca mt gen xc nh.

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- To dng nhng trnh t DNA khng m ha nm cnh cc gen m ha v ng vai tr quyt nh trong s iu ha biu hin ca gen. 2.8- Ngn hng cDNA 2.8.1- Thit k ngn hng cDNA Ngn hng cDNA l tp hp cc bn sao cDNA t tt c cc mDNA ca mt t bo. Nh vy, ngn hng c thit lp t mt loi t bo xc nh (t bo bit ha), v th, cDNA mang tnh t bo rt cao. Qu trnh chun b ngn hng cDNA bao gm cc bc sau y: - Chit tch RNA tng s, ta phi chn nhng t bo c cha nhiu mRNA cn thit v sau , lm sch mRNA, - Chn la cc mRNA cha nhiu A (polyA) bng cch tch trn ct xenlulose oligo dT, - Tng hp cDNA si kp u bng t mRNA di tc dng ca enzyme sao chp ngc nh k thut nuclease S1 hoc k thut Gubler-Hoffman, - Metyl ha cc vng hn ch (c trong on cDNA si i) c nhn bit bi enzyme EcoRI bo v vng ny khng b ct khi thao tc vi EcoRI, - To u dnh bng cch s dng on ni linker c cha trnh t nhn bit bi EcoRI, sau , ct bng enzyme EcoRI. Trong trng hp ny, ch c on ni b ct cn cDNA vn cn nguyn vn (do c metyl ha). a cDNA vo vector to DNA ti t hp, - Ghp cDNA u dnh vo vector thch hp c m EcoRI v kh nhm phosphat, - Hn kn mch nh enzyme phosphoryl ha v enzyme ligase to vector ti t hp, - a vector ti t hp vo t bo ch v nui cy trong cc iu kin thch hp to dng, - Xc nh dng cn tm v thnh lp ngn hng cDNA. Mc ch ca vic thit lp ngn hng cDNA l nhm nghin cu s biu hin ca mt gen xc nh cng vi nhng vn lin quan n s iu ha biu hin gen cng nh mi tng tc gia cc gen trong mt qu trnh sng. Ch : Khi phn tch kt qu thu c ngn hng cDNA, cn ch tc dng sinh l tng thi im th nghim. 2.8.2- Sng lc ngn hng cDNA Mt phng php ph bin nhm sng lc ngn hng cDNA c bt u t vic to canh trng nui cy cha ng nhiu khun lc t ngn hng cDNA. DNA plasmid t mi m cy vi khun gn vi mnh nh nitro-xenlulose (mng lai) s b bin tnh v lai vi mRNA tng s. Mi mRNA khc nhau s c lai trn nitroxelulose vi th t cDNA b sung ca n. Cc mRNA bm vo mng lc c tch ra v dng tng hp protein m n m ha bng h thng dch m invitro. Protein hnh thnh c em phn tch xem c phi l sn phm ca mRNA cn tm hay khng, v t , xc nh vi sinh vt nui cy cha gen mong mun. Sng lc tt c cc m nui cy theo cch trn xc nh dng n th hin gen tm kim.

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CHNG I: KHI NIM C BN V CNG NGH SINH HC (5 tit) 1.1. Khi nim c bn v cng ngh sinh hc 1.2. Cc lnh vc nghin cu ca cng ngh sinh hc 1.3. Lc s pht trin ca cng ngh sinh hc 1.4. V tr ca cng ngh gen trong cng ngh sinh hc 1.5. Khi nin v t bo gc 1.6. Thc phm chuyn gen 1.7. Mt s kha cnh v kinh t, khoa hc ca cng ngh sinh hc hin i 1.8. Cc vn php l ca cng ngh sinh hc hin i
CHNG 2: CNG NGH DNA TI T HP

2.1- Khi nim DNA ti t hp 2.2- Cc cng on chnh to DNA ti t hp 2.3- Cc phng php to DNA ti t hp 2.4- Mt s phng php a DNA ti t hp vo t bo ch 2.5- K thut tch dng (to dng) 2.6- Mt s phng php xc nh dng cn tm 2.7- Ngn hng gen (Genomic library) 2.8- Ngn hng cDNA

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