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Chng 10

i cng v Cng ngh DNA Ti t hp


Vic tinh ch enzyme ct gii hn u tin (1970) v s dng n to ra cc phn t DNA ti t hp u tin trong ng nghim (1972-1973) l nn tng cho s ra i ca k thut di truyn (genetic engineering) v cng ngh DNA ti t hp (recombinant DNA technology). Chnh s pht trin nhanh chng ca lnh vc ny khng nhng a li khi lng tri thc khng l v cu trc v c ch hot ng ca cc gene, cc b gene prokaryote v eukaryote m cn tr thnh lc lng sn xut trc tip ca x hi: to ra hng lot cc ch phm y-sinh hc hu ch t cc t bo vi khun, nm men; to cc ging sinh vt mi... gp phn gii quyt nhng vn thc tin t ra trong y hc v trong cng tc chn to ging. Trong chng ny chng ta s tm hiu cc c tnh ca enzyme ct gii hn, cc nguyn l c bn ca k thut DNA ti t hp v mt s ng dng ca lnh vc cng ngh ny.

I. Cc cng c chnh ca k thut to dng DNA ti t hp


1. Cc enzyme ct gii hn 1.1. Enzyme ct gii hn l g? Enzyme ct gii hn (restriction endonuclease) hay gi tt l enzyme gii hn (restrictase) l loi enzyme c kh nng nhn bit on trnh t nucleotide c hiu trn cc phn t DNA v ct c hai si DNA b sung ti cc v tr c th. 1.2. Vai tr ca cc enzyme ct gii hn T 1953 ngi ta pht hin thy rng, khi a DNA ca mt ni vi khun E. coli ny vo t bo thuc mt ni khc thng th DNA c a vo, gi l DNA ngoi lai hay DNA l, mt hn hot tnh di truyn v hu nh bao gi cng b phn ct thnh cc on ngn. Ch trong mt s t trng hp DNA l mi khng b phn ct v do n c th ti bn trong t bo ch. iu chng t DNA l c sa i bng cch no di s kim sot ca t bo ch. Cc hin tng ni trn xy ra ch yu khi cc th thc khun (phage) xm nhim cc t bo vi khun. Cho n u thp nin 1970 ngi ta mi bit r rng cc t bo vi khun l nhng h thng cha c hai loi enzyme: cc enzyme sa i v cc enzyme ct gii hn. Chng u c i tng nhn bit l cc on trnh t ca DNA vt ch v DNA ngoi lai, nhng c vai tr khc nhau. C th, cc enzyme sa i (methylase) ng vai tr bo v DNA vt ch

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bng cch gn thm nhm methyl (-CH3) mt s base nht nh trong on nhn bit (recognition sequence) hay on ch (target sequence). Hin tng methyl ho (methylation) ny thng xy ra i vi adenine v bin i n thnh N-6 methyladenine. Trong khi , cc enzyme gii hn li ng vai tr v hiu ho hot tnh di truyn ca cc DNA l bng cch phn ct cc v tr c th chng no n cha c sa i cho ging vi DNA vt ch. Nh vy, cc enzyme gii hn ng vai tr l hng ro bo v t nhin ca cc vi khun nhm chng li s xm nhp ca cc phage l. 1.3. Tnh cht chung ca cc enzyme gii hn Trc tin, cn lu rng cc enzyme gii hn ch pht hin thy cc vi khun m khng c cc eukaryote. V vy, tn gi ca cc enzyme gii hn thng dng l tn h thng, c biu th bng ba hoc bn ch ci vit tt ca vi khun m t enzyme c chit xut. Ch ci u tin c vit hoa ch chi (genus) v hai ch ci tip theo vit thng ch loi (species), v khi cn thit thm ch ci th t ch ni hoc chng (strain, type). Ngoi ra, phn bit cc enzyme cng mt ni ngi ta dng s La M km theo sau tn h thng (xem bng 10.1).

cc on DNA ni cc u dnh

u dnh

u dnh

+ DNA ligase

DNA ti t hp

Hnh 10.1 Enzyme gii hn (EcoRI) ct cc DNA khc nhau, nh c th kin to phn t DNA ti t hp in vitro (bng enzyme DNA ligase).

Tnh cht quan trng nht ca cc enzyme gii hn l tnh c hiu v tr, ngha l chng c th nhn bit on trnh t DNA c th ct v tr xc nh. Tu theo v tr ct so vi on nhn bit m chia ra hai loi:

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loi I bao gm cc enzyme gii hn ct bn ngoi phm vi on nhn bit v loi II bao gm cc enzyme ct c hiu bn trong on nhn bit. y chng ta ch xt cc enzyme gii hn loi II vn c xem l cng c hiu nng (ging nh con dao m tinh vi) cho php thao tc trn cc gene trong k thut DNA ti t hp (hnh 10.1). c trng ni bt ca cc on ch l c kch thc ngn, 4-8 cp base, v c tnh i xng xui ngc (palindrome). Nhn chung, cc enzyme gii hn khc nhau c hai kiu ct sau y: ct lch v ct thng. Vi kiu ct lch tc l cc v tr ct trn hai si ca DNA si kp l so le, to ra cc on DNA c cc u si n gm mt s base b sung gi l cc u dnh (cohesive/sticky ends). Cc enzyme gii hn nh th c vai tr to ln trong vic kin to DNA ti t hp in vitro (hnh 10.1). in hnh y l EcoRI v BamHI (bng 10.1). Vi kiu ct thng, tc ct cng v tr trn c hai si ca DNA si kp, do to ra cc on DNA c cc u bng (blunt ends); v d, SmaI...(bng 10.1). Cc enzyme gii hn khc nhau c on ch ging nhau, mc d v tr v kiu ct c th ging hoc khc nhau, gi l cc enzyme gii hn tng ng (isoschizomers); v d, SmaI v XmaI (bng 10.1).
Bng 10.1 Cc trnh t nhn bit v v tr ct ca cc enzyme gii hn c chn lc (mi tn ch v tr ct; cc trnh t y c ch ra trn si 5'3') Ngun vi sinh vt Arthrobacter luteus Bacillus amyloliquefaciens H Escherichia coli RY13 Haemophilus influenzae Rd Haemophilus influenzae Rd Nocardia otitidis-caviarum Providencia stuartii Serratia marcescens Sb Xanthomonas malvaccarum Tn enzyme AluI BamHI EcoRI HindII HindIII NotI PstI SmaI XmaI Trnh t nhn bit AGCT GGATCC GAATTC GTPyPuAC AAGCTT GCGGCCGC CTGCAG CCCGGG CCCGGG

2. Cc vector thng dng trong k thut di truyn 2.1. DNA ti t hp l g? DNA ti t hp (recombinant DNA) l phn t DNA c to ra trong ng nghim bng cch kt hp cc DNA t cc ngun (loi) khc nhau, theo mt quy trnh k thut nht nh, gi l k thut ti t hp DNA. Thng thng mt phn t DNA ti t hp bao gm mt phn t DNA c bn cht l plasmid hoc phage nguyn vn gi l vector (th ti) v mt on DNA t ngun khc mang mt gene hoc yu t iu ha mong

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mun c cho xen vo; n c gi l DNA ngoi lai (foreign DNA). 2.2. Hai loi vector thng dng Vector l phn t DNA c kch thc b hoc va phi, ng vai tr l vt trung gian mang truyn on DNA ngoi lai nghin cu vo trong t bo th nhn (t bo kh bin) bng con ng bin np (transformation) hoc ti np (transduction). C hai loa vector thng dng l cc plasmid hoc cc phage. Cc plasmid vi khun (hnh 10.2) c s dng rng ri hn c, bi v chng c cc c im sau: (i) c kh nng xm nhp vo t bo vt ch m vn hot ng (ti bn, biu hin gene) bnh thng; (ii) c trng lng phn t thp nn d dng tinh chit; (iii) s bn sao trong mi t bo vi khun thng kh cao; v (iv) c bit l, mt s plasmid c cha cc gene khng thuc tin li cho vic theo di v pht hin s c mt ca plasmid ti t hp trong vi khun ch.

cc khi im ti bn

Hnh 10.2 Cc plasmid c cha khi im ti bn (ori), cc im ct ca mt s retrictase v cc gene khng ampicillin (AmpR), kanamycin (KanR).

Trong s cc phage dng lm vector th phage lambda () c nhiu u th nht, bi l phn gia ca b gene c cha mt s gene khng quan trng v khng lin quan vi s ti bn ca n, nn thun li cho vic xen on DNA mong mun vo y. Cc phage khng cha cc gene khng thuc cho nn vic theo di phage ti t hp c xc nh da vo cc vt tan dng tnh (positive plaques) trn nn vi khun. 3. Thit lp phn t DNA ti t hp in vitro

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3.1. Phng php s dng cc u dnh Bt k on DNA no nu c ct bi cng mt loi enzyme gii hn (v d, EcoRI) cho cc u dnh th c th dnh lp li vi nhau v c ni bi DNA ligase (hnh 10.3). Phng php thnh lp phn t DNA ti t hp kiu ny ln u tin c a ra bi J.Mert v R.Davis nm 1972 bng thc nghim trn cc virus. V sau , ln u tin nm 1973, H.Boyer v nhm nghin cu ca S.Cohen to ra c phn t DNA ti t hp gm vector l plasmid nh pSC101 ca E. coli v DNA ''ngoi lai'' l mt plasmid khc. Chnh s kin ny t nn mng v m ra trin vng to ln cho k thut DNA ti t hp sau ny.
DNA s c xen vo

DNA c ct vi EcoRI

Cc u dnh

Ti t hp DNA + DNA ligase

Ni sai

DNA ti t hp

Ni sai

Hnh 10.3 Hai phn t DNA khc nhau c ct bi cng mt enzyme gii hn th c th ni vi nhau nh xc tc cu DNA ligase.

3.2. Phng php ni trc tip hoc tng hp cc u b sung i vi cc on DNA c to ra bng cch x l enzyme gii hn ct thng nh HindII chng hn, th vic ni cc on DNA c u bng c to ra c th thc hin theo hai cch sau: Ni trc tip bng DNA ligase ca phage T4 hoc tng hp thm cc u dnh vo cc u 3' mt s nucleotide b sung bng cch s dng cc enzyme end transferase, ri sau cc on DNA nh th s c ni vi nhau bi DNA ligase ca vi khun. C s ca phng php kt hp DNA ny c thc hin ln u tin gia DNA ca virus SV40 vi DNA ca phage bi L.Lobban v

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D.Kaiser (1972), v D.Jackson v P.Berg (1972)

II. To dng gene hay DNA ti t hp


1. Nguyn tc chung V nguyn tc, k thut DNA ti t hp hay to dng (cloning) gm cc bc chung nht nh sau: (1) Tch chit v tinh sch DNA thuc cc ngun khc nhau (gm vector v DNA mang gene mong mun); (2) to ra phn t DNA ti t hp in vitro; (3) a phn t DNA ti t hp vo trong t bo nhn, thng l E. coli hoc nm men. Hnh 10.4 m t mt quy trnh k thut n gin nh th. Tuy nhin, trn thc t, s phc tp l bc (4), pht hin v phn lp cc dng DNA ti t hp c hiu.
DNA ngoi lai Cc on ct bi enzyme gii hn Enzyme gii hn Ni cc u dnh

Plasmid ti t hp

Bin np

T bo ch E. coli

T bo c bin np

Hnh 10.4 Mt quy trnh bin np DNA ti t hp vi vector l plasmid, enzyme gii hn EcoRI, enzyme ni - DNA ligase, v t bo nhn l E. coli.

Trong t bo ch, phn t DNA ti t hp c th biu hin gene mong mun (cho sn phm protein) hoc ti bn c lp nhiu ln to ra hng lot bn sao ca n, v khi t bo ch phn chia s ko theo s to dng phn t (molecular cloning). Mt khc, do tc phn chia rt nhanh ca cc vi khun nn c th to hng triu bn sao mong mun trong mt thi gian ngn. V th nh khoa hc c th tch dng bt k mt gene no

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dng cho nghin cu hoc cho sn xut trn quy m cng nghip mt s lng ln cc protein vn l nhng ch phm y-sinh hc no . 2. Quy trnh to dng gene ti t hp By gi ta xt mt quy trnh k thut to dng m vic pht hin DNA ti t hp da trn kh nng khng thuc do vector plasmid mang li. Bc 1: Tinh chit DNA Gi s tinh chit c plasmid c cha hai gene khng ampicillin v tetracyclin, k hiu l AmpR v TetR; v cng gi thit rng gene TetR c cha im ct ca EcoRI, v phn t DNA ngi c mang gene insulin. Bc 2: Kin to phn t DNA ti t hp in vitro Trc tin, dng enzyme gii hn u dnh EcoRI (xem bng 10.1) ct vng plasmid ti gia gene TetR v cho ct DNA ngi, trong s cc on b ct c mt on mang gene insulin. Sau em trn ln hai loi DNA trn trong ng nghim vi DNA ligase. Kt qu l c th xy ra ba trng hp: (1) Plasmid t ni li thnh mch vng nh lc u; (2) on DNA t ni li thnh mch vng; v (3) Plasmid ti t hp c mang gene insulin, v c th mang mt on DNA khng phi gene . Bc 3: Bin np v pht hin dng DNA ti t hp chung a cc DNA c x l vo cc t bo E. coli. Nu phn t c kch thc ln ngi ta phi x l vi khun 'th nhn' bng chlorid calcium (CaCl2) lm cho mng tr nn thm c d dng. Sau em cy ring r cc vi khun trn mi trng c ampicillin v theo di. + Nu c xut hin khun lc (cc vi khun trong cng mt khun lc th thuc mt dng v chng bt ngun t mt vi khun ban u), chng t vi khun c mang gene AmpR, tc l chng c mang plasmid ban u (trng hp 1) hoc plasmid ti t hp (trng hp 3). Ngc li, nu ch cy khng xut hin khun lc, chng t vi khun mang DNA t ni (trng hp 2). + K , em cy ring r cc vi khun thu c sang mi trng c tetracyclin. Nu c xut hin khun lc, chng t vi khun c mang gene TetR nguyn vn (trng hp 1). Nu khng c khun lc, chng t vi khun em cy c mang DNA ti t hp (trng hp 3); v gene TetR b bt hot do on DNA xen vo. Bng cch theo di nh vy cho php xc nh c dng vi khun mang DNA ti t hp, nhng vn cha bit c u l cc dng c hiu, ngha l c mang gene insulin. Hnh 10.5 di y m t mt quy trnh n gin v to dng vi khun mang gene insulin ngi.

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gene TetR gene AmpR

DNA ngi gene insulin

cc u dnh

Lai ho + DNA ligase Bin np T bo vi khun To dng Nui cy Dng


Ch c cc vi khun cha DNA ti t hp sinh trng c

NST vi khun

t cc vi khun ln mi trng c b sung ampicillin

Tinh chit DNA

Hnh 10.5 S th nghim to dng vi khun mang DNA ti t hp, y l gene insulin ngi.

Bc 4: Chn dng DNA ti t hp c hiu V nguyn tc, trong c triu php th mi c mt t bo mang gene mong mun. Vi trng hp trn y chng hn, ngi ta c th s dng phng php min dch hc bng cch dng khng th chng li protein c sinh ra bi dng vi khun tng ng (tc huyt thanh tm gene khng insulin). Ni chung, tm dng lai c hiu ngi ta s dng cc mu d l mRNA hoc rRNA c hiu. Chng hn, trong trng hp nu cn chn dng lai mang on mRNA c th, ngi ta em cy u cc dng vi khun c cha DNA ti t hp ln trn mt thch ca hp petri cha mi trng nui cy. Sau ng du ln mng lc nitrocellulose, v thu c bn sao. Vic x l bn sao bng NaOH s lm cho cc t bo vi khun tan v ti ch (in situ), v cc DNA thot ra t chng s b bin tnh (cc si n tch ri nhau) v dnh vo mng lc. Sau em nhng

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mng lc ny vo mu mRNA tng ng c tinh khit v nh du phng x (P32); mu RNA ny c gi l vt d phng x (radioactive probe). Nu dng no c cha DNA m ho cho mRNA th s xy ra hin tng lai gia mRNA v vng si n tng ng trn DNA . Sau khi loi b cc mRNA khng lai c, ngi ta t mt ming phim nh ln trn mng lc; nhng vt nh xut hin trn nh phng x t ghi cho thy v tr ca dng mang DNA b tr vi mu RNA. T y c th tch ring cc dng lai c hiu s dng cho cc nghin cu tip theo.
Sinh trng qua m
T bo + pUC19 T bo + pUC19 T bo khng + on xen c bin np Mi trng + ampicillin + X-gal Cc khun lc trng: pUC19 + on xen Cc khun lc xanh: ch c pUC19

Cc dng khng ampicillin Vt d phng x

(a)

(b)

Mng

Cc dng chn lc Mng Phim tia X

(c)
Hnh 10.6 Xc nh cc dng vi khun mang plasmid c xen mt on DNA (gene) c hiu bng vt d phng x l mRNA - sn phm ca n.

Hnh 10.6 minh ha mt cng on ca quy trnh th nghim DNA ti t hp vi khun E. coli khi s dng mi trng nui cy c b sung ampicillin i vi ba kiu t bo: t bo c mang plasmid ti t hp, t bo ch mang plasmid pUC19 khng ti t hp, v t bo khng bin np c (hnh 10.6a). Qua m sinh trng, cc t bo no c mang plasmid ti t hp v plasmid khng ti t hp s mc thnh cc khun lc (mu sc tng ng y l trng v xanh; hnh 10.6b). Sau khi chn ra cc dng c xen plasmid ti t hp, a ln mng lc v cho tin hnh lai ha gia RNA v DNA (gene) ca n bng cc vt d phng x nh ni trn. Sau a sn phm lai phn t ny ln tm phim X quang nh v

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gene quan tm t dng ti t hp (hnh 10.6c). 3. Tng hp v to dng cDNA i vi trng hp cn cho biu hin mt gene l (sn xut mt protein) mong mun trong vi khun, ngi ta c th tng hp gene ca n da trn khun mu mRNA v enzyme phin m ngc tinh ch t cc virus RNA (xem chng 5). Sau cho xen gene ny vo plasmid, ri em cy vo vi khun v xc nh cc dng cDNA c hiu. y ta ch xt hai bc u: Bc 1: Tng hp cDNA Nh bit, cc mRNA eukaryote u c ci ui poly(A) u 3'. Chnh trnh t ny to iu kin thun li cho vic tng hp si DNA b sung, cDNA. Khi em trn ln cc on ngn gm cc nucleotide thymine (oligodT) vi mRNA ny s xy ra s lai ho gia n vi vng ui mRNA. on oligo(dT) lm mi cho enzyme phin m ngc (reverse transcriptase) tng hp si cDNA m sn phm l si kp lai RNAcDNA. u 3' ca si cDNA c tng hp c ci 'chp' (tng t u 5' ca mRNA). Tip theo, bng cch x l vi NaOH, si mRNA b loi ra; k ci 'chp' u 3' ca cDNA li lm mi cho DNA polymerase I tng hp si th hai dc theo si khun vn c ca n. Sn phm cDNA by gi cn mang ci 'vng' si n. Sau , ci vng ny c ct b bng cch x l vi nuclease S1 to ra cDNA si kp. Bc 2: Xen on cDNA vo plasmid xen on cDNA vo plasmid ngi ta c th dng enzyme end transferase gn thm ''ui'' homopolymer (v d, CCCC....) vo cc u 3' ca cDNA. V plasmid sau khi c m vng, cng phi lp thm u 3' nhng trnh t tng ng l GGGG... cng vi enzyme trn. Tuy nhin, cch ph bin hn c l gn thm vo c hai 'u bng' ca si kp cDNA ny bng cc oligonucleotide gm 8-10 cp base, nh xc tc ca DNA ligase T4. Sau , dng enzyme gii hn thch hp ct ''on ni'' ny to ra cc u dnh. ng thi ct plasmid bi cng mt enzyme ti gene TetR. Hai DNA ni trn c ni vi nhau bng DNA ligase to ra plasmid lai.

III. Cc phng php biu hin cc gene c to dng


Mt s cc vector c s dng trong cc th nghim to dng ti t hp (nh cp) vn c th c s dng nh l nhng vector biu hin (expression vectors). Cc vector ny c th sn sinh ra cc sn phm protein ca cc gene c to dng. Chng hn, cc vector pUC v pBS c xen vo DNA di s kim sot ca lac promoter, vn nm pha

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trc so vi v tr to dng phc (multiple cloning site). Nu nh mt on DNA c cho xen c mt trong cng khung c m th n lm gin on gene lacZ', s sinh ra mt protein dung hp (fusion protein). N s c mt phn trong trnh t ca protein beta-galacyosidase ti u amin v trnh t protein khc na vn c m ha trong DNA c xen vo, u carboxyl ca n. Tuy nhin, nu ta quan tm ti s biu hin cao ca vector c to dng, th cc vector chuyn biu hin thng hot ng tt hn. C hai yu t in hnh cn thit cho s biu hin gene c hot tnh: mt promoter mnh v mt v tr bm ca ribosome m bao gm lun c trnh t Shine-Dalgarno nm gn codon khi u AUG. Trn thc t, ngi ta s dng cc vector biu hin c cc promoter mnh (expression vector with strong promoters), chng hn nh promoter ca operon tryptophan. N to thnh c s cho nhiu vector biu hin k c ptrpL1. Ngoi ra, ngi ta cn s dng cc vector biu hin dng cm ng (inducible expression vectors). Trng hp ny thng tin li ch, n gi cho mt gene c to dng trng thi ng cho ti khi ta sn sng cho n biu hin. Mt l do l ch, cc protein ca eukaryote c sn sinh mt s lng ln vi khun c th gy c. Ngay c cc protein vn khng c thc s, chng cng c th c to ra nhiu n mc gy ri lon s sinh trng ca vi khun... Promoter ca operon lactose (lac promoter) l vector biu hin kiu cm ng n mt mc no , c l l vn gi bt hot cho ti khi c kch hot bi cht cm ng allolactose hoc bng cht tng hp tng t ca n l IPTG. Tuy nhin, s biu hin vn km bi cht c ch lac l khng y hon ton, v s biu hin no ca gene c to dng vn c th pht hin c ngay c khi khng c mt cht cm ng. Mt cch xoay quanh vn ny l cho biu hin gene mong mun trong mt plasmid hay phagemid m n mang c gene lacI ca ring n, nh l plasmid pBS chng hn. By gi chng ta th tm hiu mt phng php sn xut insulin ngi bng con ng tng hp DNA v cho biu hin gen E. coli. Trc tin cn lu rng, thc hin c iu ny ngi ta phi da trn thnh tu mi nht t vic nghin cu cu trc chi tit v qu trnh tng hp cc ch tit insulin t tuyn tu vo mu. Ni vn tt th sn phm s cp ca qu trnh dch m t phn t mRNA hon chnh l preproinsulin gm on peptide "tn hiu dn u" v cht tin thn ca insulin l proinsulin; on pre- b tch b trong qu trnh tng hp. Proinsulin c ch tit l phn t gm ba on A, B v C lin nhau trong mt cu trc "hnh quai" c ba cu disulfur; khi on peptid C (33 amino acid) b ct b bi enzyme

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c th trong cc ti ca t bo tuyn ty s to ra cc sn phm insulin c hot tnh. Phn t insulin gm hai chui polypeptid A (21aa) v B (30aa) ring bit c duy tr cng nhau bi hai cu disulfur. T y ta c th hnh dung qu trnh tng hp gene insulin nhn to (cDNA) v cho sn xut hormone ny E. coli nh sau. Trc tin, dng mRNA ca proinsulin lm khun tng hp y mt DNA si kp bng con ng phin m ngc nh trnh by trc. Sau lp thm b ba khi u ATG nhn to (m ho amino acid u chui polipeptide) vo u 5' bng phng php ho hc. Tip n, cho n kt hp vi mt phn ca operon lactose (gm mt on ca gene galactosidase v ton b promoter) ca E. coli n c th hot ng c trong t bo th nhn. Sau gene "lai" ny c xen vo plasmid pBR322 ( y khng i su vo cc chi tit k thut, chng hn nh s dng cc on ni, linker, v cc enzyme gii hn). Khi a plasmid lai ny vo vi khun E. coli, n s sn sinh ra cc protein lai gm mt on peptide ca -galactosidase ni lin vi phn t proinsulin qua gc methionine (MET). Sau khi phn lp protein ny v x l in vitro bng cyanogen bromide th gc MET b ct b (ko theo phn enzyme ca vi khun l -galactosidase tch ra) v thu c proinsulin nguyn vn. Cui cng, nh x l vi enzyme thch hp, on peptid C gia proinsulin c tch ra v c th thu c insulin dng tinh khit. Cng cn lu rng, hin nay ngi ta to ra c cc dng vi khun v cc chng nm men mi c hiu c kh nng sn xut insulin trn quy m cng nghip vi ch s insulin cao, v c bit l, cc chng ny c th trc tip bi xut sn phm c hiu vo mi trng nui cy. Trong trng hp , cc t bo chuyn sn xut insulin vn c duy tr v ti s dng vi hiu qu kinh t cao.

IV. ng dng ca cng ngh DNA ti t hp


1. Cng ngh DNA ti t hp vi vic nghin cu b gene Vic tch dng ti t hp cho php nhn c mt s lng ln bt k gene hoc vng iu ho no tin hnh phn tch trnh t nucleotide, xc nh cc vng chc nng v ch ra cc c ch hot ng ca chng. Nh a li nhng hiu bit mi v t chc v hot ng ca cc b gene prokaryote (nh cc khi im ti bn, cc vng iu ho phin m, cc gene nhy v.v.) v cc b gene eukaryote (nh cc centromere, telomere, cc gene phn on, cc gene gi, DNA lp li v.v.) nh c tho lun cc chng trc.

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DNA ngi

vector YAC

DNA cn to dng v DNA plasmid c ct bi cng enzyme gii hn

ct tng phn u dnh cc on NST ln u dnh

Cc plasmid cha cc on xen khc nhau

u dnh Ti t hp + DNA ligase Nhim sc th nm men nhn to xen DNA ngi

vi khun c bin np vi hn hp hoc cc plasmid nui cy vi khun ch mi dng l mt 'volume' trong th vin gene

lln ti 1.000.000 bp Bin np vo t bo nm men

Dng

(a) (b) Hnh 10.7 Xy dng th vin gene hay th vin b gene (gene or genomic library) bng cc th nghim to dng plasmid ti t hp vi khun (a) v s dng nhim sc th nm men nhn to, YAC (b).

Bng cc th nghim to dng plasmid ti t hp kinh in vi khun E. coli, v bng cch ci tin s dng nhim sc th nm men nhn to ny (yeast artificial chromosome = YAC; hnh 10.7), ngi ta thnh lp cc th vin gene (gene library) hay th vin b gene (genomic library) trn c s tin hnh lp bn vt l (physical map) v xc nh trnh t DNA b gene ca nhiu sinh vt khc nhau, k c b gene ngi (NHGRI 2005). Nu nh vo nm 1977, F.Sanger xc nh y cc trnh t phage X174 (5386 nucleotide vi 9 gene) v phage G4 (5577 cp nucleotide vi 10 gene), th n nay ngi ta xc nh v lp bn cho cc DNA c kch thc ln hn nhiu; v d: b gene phage lambda gm 48.502 cp base vi khong 61 gene (1983), nhim sc th s 3 ca nm men Saccharomyces cerevisiae gm 370.000 cp base (1992) v.v... ng k l, D n B gene Ngi (Human Genome Project = HGP; hnh 10.8) chnh thc i vo hot ng t 1990 do James Watson ch tr cng vi s cng tc ca nhiu nh khoa hc trn th gii. Vic phn tch trnh t b gene ngi c hon thnh vo 4/2003, cho thy b

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gene (genome) ca chng ta c trnh t y gm 3.164.700.000 cp base, cha ng khong 25.000 gene m ha protein chu trch nhim xy dng nn mt b protein (proteome) gm khong 50.000 protein khc nhau trong cc t bo. HGP thc s l mt trong nhng k cng thm him v i nht trong lch s; ln u tin HGP cho php chng ta c c ton b thng tin di truyn ca t nhin lm nn con ngi (NHGRI 2005).

Hnh 10.8 Biu tng ca HGP cho thy tc ng ca d n ny ln nhiu lnh vc quan trng ca kinh t - x hi, cc vn v mi sinh, sc kho v i sng con ngi trn phm vi ton cu.

2. Cng ngh DNA ti t hp vi y-dc hc 2.1. Sn xut cc ch phm y-sinh hc bng cng ngh DNA ti t hp Nu nh vo nm 1972, Gio s Roger Guillemin (France) phi dng ti 500.000 no cu mi tinh chit c vi miligram somatostatin, mt hormone sinh trng ca vng di i th (vi cng trnh ny ng c trao gii Nobel nm 1980), th vo 10/1977 Hebert Boyer (USA) ch to thnh cng hormone ny t E. coli bng phng php DNA ti t hp. n 8/1978 chnh Boyer li l ngi u tin cho sn xut thnh cng insulin ngi t E. coli (hnh 10.9). Cc thnh tu u tin ny m ra mt k nguyn mi pht trin rc r nht trong lch s cng ngh sinh hc, cng ngh DNA ti t hp. Sau l hng lot cc ch phm khc nh cc hormone tng trng ca ngi (human growth hormone = HGH), b (BGH), ln (PGH), cc vaccine v cc interferon phng chng cc cn bnh nan ngi y nh ung th, vim gan v.v. v mt s bnh quan trng gia sc nh hin tng 'l mm-long mng' do virus gy ra... tt c u c sn xut t E. coli. c bit l, s ra i ca cc hng, cc cng ty ln u t mnh m cho s pht trin ca k ngh ny. Nh vy gp phn gii quyt mt cch c hiu qu nhiu vn thc tin t ra trong y hc, trong chn nui-th y, v nng nghip ni chung ...

272

nc ta cng c mt s cng trnh nghin cu v cc vn ny, chng hn nh nghin cu s sai khc di truyn gene hormon sinh trng ca mt s ging g Vit Nam. Qua cho thy intron I ca gene hormon sinh trng cc ging g Ri, g Ma, g c, g H di hn kch thc d on ca gene hormone sinh trng g c Tanaka cng b nm 1992 (Trn Xun Hon 2004).
Chuyn gene insulin enzyme gii hn cDNA ngi on ni Plasmid ti t hp

To dng gene insulin vi khun

chuyn gene

insulin insulin

insulin insulin

(a) (b) Hnh 10.9 (a) H. Boyer (tri) v S. Cohen; v (b) s th nghim to dng v biu hin gene insulin ngi vi khun E. coli.

Bn cnh vic sn xut cc hormone, vaccine... ni trn, ngi ta cn sn xut c cc khng th n dng (monocloning antibodies) dng : (i) xc nh vi khun gy bnh (nh thng hn chng hn); (ii) xc nh mc hormone t nh gi chc nng tuyn ni tit hoc s thay i trong qu trnh tng hp hormone do khi u gy ra; (iii) pht hin mt s protein c ngha trong chn on khi u hoc mt s tnh trng trc khi sinh; (iv) pht hin cc thuc b cm c trong mu, hoc kim tra nng thuc trong mu v t chc nhm m bo liu thuc s dng sao cho khng vt qu ngng gy c v.v. Nh c tnh c hiu v chnh xc cao v s dng d dng, vic sn xut cc khng th n dng to nn mt nhnh pht trin mau l nht ca cng ngh sinh hc, v cng vi vic sn xut cc vaccine chng tr thnh nhng phng tin quan trng nht trong chnh sch y t cng ng ca cc nc ang pht trin v xt v lu di, vic ng dng cc khng th n dng c trin vng cha c nhiu bnh trong c cc khi u c tnh. 2.2. ng dng k thut di truyn trong chn on v iu tr gene Trong chn on bnh mc phn t, thnh tu ni bt nht l vo nm 1981, ln u tin bnh thiu mu hng cu hnh lim c chn

273

on trc sinh mc gene nh phn tch DNA bng enzyme gii hn. T y m ra hai hng chnh trong chn on gene l: chn on cc bt thng bm sinh v chn on cc bt thng DNA soma. Mt s thnh tu khc t c theo hng u gm c: bnh hemophilia A, ri lon dng c Duchenne, hi chng X-fragile, bnh retinoblastoma - mt dng ung th vng mc...Theo hng sau, ngi ta p dng i vi mt s dng ung th mu nh cc lympho Burkitt, lympho nang, bnh bch cu...ng k l gn y, ngi ta xc nh c nhiu gene gy bnh quan trng ngi, nh: gene gy bnh ha x nang (cystic fibrosis) nm 1990, gene gy bnh Huntington nm 1993... Liu php gene (gene therapy) cng l mt phng thc sn xut v iu tr mi bng cc phn t tr liu. y l hng c nhiu trin vng nht nhng kh thc hin nht v n c lin quan n c vn phng php lun ln kha cnh o l. S kin ni bt nht l vo nm 1990-91, W.F.Anderson, R.M.Blaese v Ken Cuver thc hin thnh cng ca liu php gene u tin trn mt b gi bn tui mc bnh suy gim min dch phi hp (SCID) vi s thiu ht adenosine deaminase (ADA), mt enzyme cn thit cho sn xut cc khng th trong cc t bo h mim dch, gi l hi chng "bubble-boy" (bnh do gene gn u mt vai ngn nhim sc th s 20 gy ra). Mt khc, liu php gene cng m ra nhng trin vng to ln trong cha tr cc cn bnh ph bin nht, tc ng ti hng triu triu ngi trn hnh tinh nh: ung th, SIDA/AIDS, vim gan do virus, cc bnh tim mch, cc bnh thoi ha thn kinh (bnh Parkinson, bnh Huntington, bnh Alzheimer...) hay c nhng bnh mn tnh nh a thp khp. 2.3. K thut di truyn vi hnh php hc v mt s vn x hi khc K thut di truyn cn c ng dng rt hiu qu trong cc ngnh hnh php hc, chng hn bng cch s dng k thut 'du vn DNA' (DNA fingerprinting) thay cho 'du vn tay' trc y cho php cc ngnh cnh st hnh s xc nh chnh xc cc ti phm hoc nhn dng xc nn nhn trong chin tranh hoc do tai nn gy ra (hnh 10.10).
Hnh 10.10 Du vn DNA (DNA fingerprinting) c th gip cc nh nghin cu xc nh kh nghi trong trng hp ti phm. Kiu vch ngang biu th bn cht di truyn ca mt ngi. mu ny cho thy cc bng ca mu ngi mang m s S2 kh nghi trng khp vi bng chng, mu mu E(vs).

274

nc ta, trong thi gian gn y, k thut ny v ang c p dng mt cch hiu qu trong ngnh hnh s. Bn cnh c cng trnh phn tch DNA nhn din t bo trn 103 ngi Vit Kinh bng k thut PCR - SSO (Kit Innolipa). Qua phn tch so snh khong cch gene hc gia ngi Vit Kinh v 11 sc tc chu v i dng khc, kt lun rng ngi Vit Kinh gn vi ngi Thi ri n ngi Manchu (V Triu An, 1999). 3. K thut di truyn vi cc sinh vt bin i gene (genetically modified organisms = GMOs) i vi ngnh chn nui, cng ngh sinh hc ni chung v cng ngh sinh hc ni ring t nhiu thnh tu ng k, chng hn cc k thut chuyn ghp gene p dng cho hp t v phi cc gia sc nhm tng cng kh nng chng bnh v ci thin ging ni chung; cng nh cc k thut mi trong xc nh gii tnh ca phi... Hnh 10.11 cho thy kh nng ng dng k thut cy ghp gene trn trng chut c th tinh. Sau em phi ghp gene cy vo trong t cung ca con chut lm m khc. Kt qu l to ra c chut con c b lng dng khm nh mong mun. in hnh cho cc th nghim truyn gene ng vt l vo nm 1982,R.D.Palmiter, R.L.Brinster v cc ng s i hc Seatle (Philadelphia, USA) bng cch truyn gene xc nh hormone sinh trng ca chut cng vo trng th tnh ca chut bnh thng, ri cy tr li cc t bo
Tinh chit gene Tim

cc trng chut

Phi c cy trong t cung ca chut m thay th

i con

Hnh 10.11 M hnh tng qut v th nghim truyn gene ng vt (xem gii thch trong bi).

c bin i gene vo vi trng ca cc chut ci c th mang thai cho n cng. V kt qu l, cc tc gi ny thu c dng chut nht c kch thc ln gp 2-3 ln chut bnh thng, gi l chut khng l. i vi trng trt, vic s dng cc phng php chuyn ghp gene t c nhiu thnh tu to ln. Chng hn, hng Biogen (USA) nm

275

1984 chuyn thnh cng plasmid Ti vo t bo thc vt; hng Calgene v Phytogene (USA, 1984) ghp thnh cng gene khng glyphosate bo v cy bng; nm 1985 hng Molecular Genetics (USA) to c ging ng mi cho nhiu tryptophan. Nm 1993, bng k thut sng bn gene vo t bo thc vt ngi ta a c gene sn xut protein dit su vo cy ng, v kt qu l to ra c ging ng chng chu cao i vi su c thn. iu th v l vic tch cc gene c nh m, gene Nif (Nif = nitrogene fixation) t cc vi khun nt sn cy h u v a vo b gene ca cc cy trng khc to ra cc ging cy trng mi c kh nng c nh nit v cho nng sut cao. Trong chn ging vi sinh vt, ngi ta thc hin thnh cng vic chuyn gene cellulase vo vi khun (J.P.Aubert, France 1/1983), ci bin E. coli sn xut L-aspartat (hng Tanabe, Japan 1985), ghp gene vo x khun S. violacconiger ci tin vic sn sinh enzyme glucoisomerase (hng Roquette v Cayla, France 1985), ngoi ra cn to c cc ging vi sinh vt bin i gene c kh nng n cn du dng trong x l cc ph thi c c t nhm bo v mi sinh. Bn cnh vic to ra ging nm men mi c th git cht cc vi khun xut hin trong bia (hng Suntory, 1985), cn to c chng nm men sn xut insulin v interferon (A. Kimura, Japan 1986) v.v. Nhn thc r ngha v tm quan trng ca cng ngh DNA ti t hp v cng ngh sinh hc ni chung i vi s pht trin kinh t-x hi t nc ta trong th k XXI, Chnh ph ra Ngh quyt 18/CP ngy 11/4/1994 v "Phng hng pht trin ca cng ngh sinh hc Vit Nam n nm 2010". y c xem l mt bc ngot quan trng cho s pht trin ca cng ngh sinh hc nc nh trong thi gian qua v sp ti.

Cu hi v Bi tp
1. Th no l enzyme gii hn? Chng c nhng tnh cht no m c coi l cng c thit yu i vi cng ngh DNA ti t hp? Bng hai v d v enzyme gii hn, hy chng t rng t nht c hai phng php thnh lp cc phn t DNA ti t hp in vitro. 2. T cc enzyme gii hn BamHI, EcoRI v HindIII Bng 10.1 v BglII c on ch c bit l AGATCT, hy cho bit cp enzyme no s to ra cc u dnh hay u b sung (sticky/complementary ends) tng thch? Trnh t ca u dnh tng thch ny l g? 3. Gi s bn ct hai DNA khc nhau, mt vi BamHI v mt vi BglII, sau ni chng li vi nhau thng qua cc u dnh tng thch.

276

Mt khi khu ni ri, bn c th tch hai DNA ny ln na bng mt trong hai enzyme gii hn hay khng? Ti sao, hoc ti sao khng? 4. Gi s bn bin np mt DNA ti t hp cho mt vi khun v do nhm ln, bn t cc t bo c bin np trn mi trng khng c cht khng sinh no c. Kt qu m bn quan st c l g? Ti sao? 5. Gi s rng bn chit xut c mt enzyme ct gii hn t loi vi khun c tn khoa hc l Xenobacterium giganticus. Theo danh php hc, bn s vit tn enzyme nh th no? 6. Cho bit trnh t ca mt on DNA c cha mt v tr nhn bit i xng xui ngc (palindromic recognition site) cho mt enzyme gii hn l GACGATATCAACT. Hy tm trnh t ca v tr nhn bit . 7. Th no l phn t DNA ti t hp, vector tch dng? Hy nu cc bc ca quy trnh to dng gene ti t hp v cho s minh ho. 8. Gi s tinh ch c plasmid pBR322 v phn t DNA ngi c cha mt gene cn nghin cu. Hy phn tch k thut to dng gene ni trn E. coli. 9. Enzyme phin m ngc l g? N c tinh ch t loi sinh vt no v c ng dng vo khu no trong cng ngh DNA ti t hp? Gii thch v cho s minh ho. 10. C th s dng cc cht khng sinh xc nh xem liu plasmid pBR322 nhn c mt on xen trong v tr EcoRI ca n hay khng? Ti sao, hoc ti sao khng?

Ti liu Tham kho


Ting Vit Trn Xun Hon. 2004. Nghin cu s sai khc di truyn gene hormone sinh trng ca mt s ging g Vit Nam. Lun n Tin s Di truyn hc, Th vin Quc gia, H Ni. L nh Lng. 2001. Nguyn l K thut Di truyn. NXB Khoa hc v K thut, H Ni. Phan C Nhn. 1999. Cng ngh DNA ti t hp. Trong: Di truyn hc tp II (Phan C Nhn, ch bin). Trang: 257-303. NXB Gio Dc, H Ni. Hong Trng Phn. 1995. Mt s vn v Di truyn hc hin i (Ti liu BDTX cho gio vin THPT chu k 1993-1996). Trng HSP Hu. Hong Trng Phn. 1997. Di truyn hc Phn t. NXB Gio Dc. Hong Trng Phn. 1999. "Gii thiu cng ngh sinh hc"; v "C s

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khoa hc ca cng ngh sinh hc". Trong: Chuyn Cng ngh Sinh hc (Ti liu BDTX gio vin THPT chu k 1997-2000; bin son chung vi Nguyn B Lc v Bin Vn Minh). Trang: 56-96. Trng HSP Hu.

Ting Anh
Vu Trieu An 1999. Mitochondrial DNA polymorphism in the Vietnamese population. Eur. J. of Immunogenetics, 26: 471-422. Campbell NA, Reece JB. 2001. Essential Biology. Benjamin/Cummings, an imprint of Addison Wesley Longman, Inc, San Francisco, CA. Collins FS, Green ED, Guttmacher AE DNA Guyer MS. 2003. A Vision for the Future of Genomics Research. Nature, Vol.422, No.6934, p.835-847. Hartwell, Hood, Goldberg, Reynolds, Silver, Veres. 2004. Genetics From Genes to Genomes. 2nd Edition. McGraw Hill, Inc., New York. Lewis R. 2003. Human Genetics: Concepts DNA Applications. 5th ed, McGraw-Hill, Inc, NY. Palladino MA. 2002. Understanding the Human Genome Project. Benjamin/Cummings Publishing Company, Inc, Menlo Park, CA. Watson JD, Hopkins NH, Roberts JW, Steitz JA, Weiner AM. 1987. Molecular Biology of the Gene. 4th ed, Benjamin/Cummings Publishing Company, Inc, Menlo Park, CA. Watson JD, Gilman M, Witkowski J, Zoller M. 1992. Recombinant DNA. 2nd ed, Scientific American Books, New York. Weaver RF, Hedrick PW. 1997. Genetics. 3rd ed, McGraw-Hill Companies, Inc. Wm.C.Browm Publishers, Dubuque, IA. Mt s trang web Agricultural Biotechnology http://www.aphis.usda_gov/biotechnology/ http://www.agbioworld.org Online Mendelian Inheritance in Man (OMIMTM): http://www.ncbi.nlm.nih.gov/Omim/mimstats.html http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM National Human Genome Research Institute (NHGRI): http://www.ncbi.nlm.nih.gov/genome/guide/human The Institute for Genomic Research: http://www.tigr.org Celera Genomics Corp.: http://www.celera.com

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