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  • TS. CHU HONG MU

    C S V PHNG PHP SINH HC PHN T

    NH XUT BN I HC S PHM

  • Li ni u.................................................................................................................................4

    Chng 1: H GENE..............................................................................................................5

    1. KHI NIM H GENE (GENOME) ..................................................................5 2. AXIT NUCLEIC ..................................................................................................9 3. ADN V TI BN ADN..................................................................................11 4. ARN V C CH PHIN M .........................................................................19 THO LUN...........................................................................................................23

    Chng 2: C IM CU TRC GENE CA SINH VT PROKARYOT V EUKARYOT ...........................................................................................................25

    1. C IM CU TRC GENE PROKARYOT ..........................................25 2. CU TRC PHN ON GENE EUKARYOT .........................................25 3. MT S TRNH T ADN................................................................................27 THO LUN...........................................................................................................32

    Chng 3: MI LIN H GIA ADN, ARN, PROTEIN................................................33

    1. THNG TIN DI TRUYN V MT M DI TRUYN .................................33 2. PROTEIN ...........................................................................................................39 3. QU TRNH SINH TNG HP PROTEIN.....................................................42 4. IU HO BIU HIN GENE ........................................................................45

    THO LUN......................................................................................................................49

    Chng 4: ENZYME S DNG TRONG K THUT SINH HC PHN T ............50

    1. ENYME GII HN (RESTRICTION ENZYME HOC ENZYME CT HN CH (RESTRICTION ENDONUCLEASE - RE) .............................................50

    2. CC NHM ENZYME KHC.........................................................................57 3. ENZYME NUCLEASE......................................................................................60

    THO LUN......................................................................................................................61

    Chng 5: XC NH MI LIN QUAN GIA PROTEIN V C TNH SINH VT ...............................................................................................................62

    1. MT S LOI PROTEIN CHC NNG........................................................62 2. QUY TRNH XC NH CH TH PHN T PROTEIN..............................66 3. K THUT PHN TCH PROTEIN .................................................................67 4. K THUT PHN TCH ENZYME..................................................................74 5. NGHIN CU RIPS BNG WESTERN BLOT ..............................................77

    THO LUN......................................................................................................................78

    Chng 6: K THUT SINH HC PHN T TRONG PHN TCH H GENE SINH VT..........................................................................................................................79

    1. PHNG PHP TCH CHIT ADN..............................................................79 2. LAI PHN T ...................................................................................................83 3. XC NH TRNH T NUCLEOTIT CA ADN ..........................................85 4. RFLP TECHNOLOGY (K thut phn tch hin tng a hnh ca di cc

    phn on ADN).................................................................................................87

    2

  • THO LUN......................................................................................................................91

    Chng 7: PHN NG CHUI POLIMERASE (POLIMERASE CHAIN REACTION - PCR)................................................................................................92

    1. PHN NG CHUI POLIMERASE (PCR) ....................................................92 2. PHN TCH TNH A HNH CA ADN C NHN BN NGU

    NHIN (RANDQM AMPLIFIED PQLIMORPHIC DAN - RAPD) ................99 4. PHN TCH TNH A HNH CHIU DI CC PHN ON ADN C

    NHN BN C CHN LC .........................................................................106 THO LUN.........................................................................................................108

    Chng 8: T BO CH V VECTOR ..........................................................................109

    1. T BO CH ..................................................................................................109 2. VECTOR ..........................................................................................................111 3. PHNG PHP CHUYN VECTOR TI T HP VO T BO CH ..122 THO LUN.........................................................................................................125

    Chng 9: PHN LP GENE TCH DNG PHN T V BIU HIN GENE......126

    1. PHN LP GENE ...........................................................................................126 2. TCH DNG GENE .......................................................................................127 3. BIU HIN GENE...........................................................................................139 THO LUN.........................................................................................................151 Ti liu tham kho chnh .....................................................................................152 nhng cng trnh ca tc gi cng cc cng tc vin cng b.....................155

    C S V PHNG PHP SINH HC PHN T .....................................................162

    3

  • Li ni u Sinh hc phn t hin i ang pht trin mnh v tr thnh nng ct ca Cng ngh sinh hc. Vit Nam, thnh tu nghin cu Sinh hc phn t v p dng k thut phn t trong nghin cu Khoa hc s sngv Cng ngh sinh hc c nhiu ng gg trong vic chm sc, bo v sc kho con ngi; trong nh gi ti nguyn sinh vt; trong chn ging v sn xut nng lm ng nghip. Sinh hc phn t l mn Sinh hc hin i, c ging dy nhiu trng i hc v ang c nhng ng gp nht nh trong o to lp ngi c tri thc v Cng ngh sinh hc gp phn vo s nghip Cng nghip ho, hin i ho C s v phng php Sinh hc phn t c bin son t nhiu ti liu, bi ging v cng trnh nghim cu mi v Sinh hc phn t hin i ca cc tc gi trong v ngoi nc, nhm cung cp nhng kin thc c bn v nguyn l v ng dng ca Sinh hc phn t, lm ti liu cho nghin cu, ging dy v hc tp mn ny trng i hc. Cun C s v phng php Sinh hc phn t c cu trc bi 9 chng: Chng 1. H gene

    Chng 2. c im cu trc gene ca sinh vt Prokaryot v Eukryot Chng 3. Mi lin h gia ADN, ARN, Protein

    Chng 4. Enzyme s dng trong k thut sinh hc phn t Chng 5. Xc nh mi lin quan gia protein v c tnh sinh vt Chng 6. K thut sinh hc phn t trong tch h gene sinh vt Chng 7. Phn ng chui polimerase (PCR) Chng 8. T bo ch v Vector Chng 9. Phn lp gene, tch dng phn t v biu hin gene.

    Tc gi trn trng cm n PGS.TS. Nguyn Trng Lng, TS. Lng Th Hng Vn c v gp cho bn tho, xin cm n nhng ng gp ca Hi ng nghim thu nh gi gio trnh ca trng i hc S phm - i hc Thi Nguyn v cm n cc kin ng gp qu bu ca ng o cc nh khoa hc. Trong qu trnh bin son chc chn c nhng sai st, tc gi rt mong nhn c nhng gp ca bn c. Mi ng gp xin gi v Khoa Sinh - K thut Nng nghip trng i hc S phm - i hc Thi Nguyn.

    Tc gi

    4

  • Chng 1: H GENE Tm tt: S ra i ca sinh hc phn t c nh du bng thi im m Oatsn v Cric (1953) pht hin ra cu trc ADN. Tri qua hn 40 nm (1953 - 2000) sinh hc phn t t c nhng thnh tu v i m nh cao ca s pht trin ny l nhng khm ph bn cht sinh hc ca s sng cp phn t v xy dng cc k thut sinh hc phn t ng dng vo thc tin. Geneomics v Proteomics l vn ang c c bit quan tm hin nay m c s ca cc lnh vc ny l nhng pht hin v cu trc v chc nng ca axit nucleic, v c im ca genome nhn, genome ti th, genome lp th. Nhng im khc nhau v cu trc v chc nng ca cc h gene cho php ng dng vo thc t chn ging v nghin cu ngi. Cng vi cu trc ADN v ARN cn c c im ca qu trnh ti bn ADN v phin m cng c quan tm, v n l c s ca nhng k thut sinh hc phn t - cc thao tc ADN v ARN. Ni dung c bn ca chng cp n nhng c s ca sinh hc phn t. Ni dung ca chng gm 4 vn c bn: (1). Khi nim h gene; (2). Axit nucleic; (3). ADN v ti bn ADN, (4). ARN v c ch phn m.

    1. KHI NIM H GENE (GENOME) Qu trnh sinh trng v pht trin ca sinh vt tri qua rt nhiu giai on v tt c qu trnh u ph thuc vo s iu khin ca cc gene. Cu trc, chc nng ca t bo c quyt nh trc tip bi protein, l sn phm cui cng ca s biu hin gene. Qu trnh th hin hot ng ca gene qua protein, b nh hng rt ln bi cc yu t ngoi cnh nh nh sng, ch dinh dng, s cng sinh v s tng tc ca cc gene trong h gene ca t bo. Nh vy, gia cc thnh phn ca h gene trong t bo sng c s tng tc vi nhau v c mi quan h vi cc yu t ca mi trng. Trong t bo, bn cnh genome trong nhn, h thng di truyn cn phn b trong lc lp, ti th v plasmid (vi khun). Cc c quan t ny cng tham gia vo qu trnh sinh tng hp protein ca t bo. T bo l n v cu trc v chc nng c bn ca cc c th sng, t bo cn l n v ca s di truyn. H gene l ton h cc gene trong t bo ca c th sinh vt, h gene ha ton b thng tin di truyn c trng cho tng loi, cho tng c th trong loi. sinh vt Prokaryot h gene gm ton b cc gene trong t bo; cn Eukaryot

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  • h gene gm ton b cc gene trong t bo n bi (n). T bo n bi c mt h gene, cc sinh vt hoc t bo lng bi c hai h gene, sinh vt a bi c nhiu h gene. H gene ca sinh vt Eukaryot bao gm h gene nhn (genome nhn), h gene ti th (genome ti th), h gene lc lp (genome lc lp). H gene Prokaryot ch c mt thnh phn gm cc on ADN khng ging nhau; cn h gene Eukaryot c ba thnh phn ADN khng ging nhau: Cc on ADN lp li nhiu (chim khong 25%), cc on ADN lp li t (30%) v cc on ADN khng lp li (45%). 1.1. H gene nhn H gene nhn l genome ln nht trong t bo xt v mt khi lng cng nh s lng gene m ho. ADN nhn c sp xp gn trong nhim sc th trong s lin kt vi protein cha histone v khng cha histone. ADN c vai tr m ho thng tin di truyn, cn protein th bo v v tham gia iu khin s sao chp, phin m c chnh xc. Qu trnh c bn trong pht trin ng, thc vt l s nhn i vt cht di truyn v phn u cho cc t bo con khi t bo phn chia. T bo thc vt cha mt lng ln ADN, khi lng ny thay i theo loi. Arabidosis thaliana c lng ADN nh nht (0.07 picogram). Allium cepa c lng ADN nhiu hn 33,5 pg/ haploid genome (h gene n bi). 1.2. H gene lc lp Cho n nm 1962, ngi ta mi khm ph ra ADN v ribosom c trong lc lp. Nh vy, lc lp hoc trong lp th cha tt c b my cn thit t mnh biu hin hot ng gene. H gene lc lp l ton b cc gene trong lc lp hay ton b lng thng tin di truyn cha trong ADN ca lc lp. Lng ADN trong lc lp rt ln, chim ti 15% tng lng ADN c th thc vt, ribosom chim 60% lng ribosom ca t bo, ADN lc lp c cu to bi cc phn t c cu trc mch vng, c trng lng phn t 83 - 128 x 106, trong gn 85% phn t ADN l mch n. ADN lc lp lm khun phin m tng hp mARN chloroplast. y cc phn t mARN ch c khong 20 nucleotit, chng thc hin s tng hp protein ti ch trong chloroplast. Ribosom trong chloroplast l 70S bao gm 30S v 50S. Sau khi tng hp xong protein c vn chuyn n ni m lc lp cn thit. V c bn h thng di truyn trong lc lp tng t vi h thng di truyn ca sinh vt Prokaryot. Do vy c nhiu cuc tranh lun, c phi vt liu di truyn ca sinh vt tin nhn l ngun gc ca ADN lc lp? Ngoi ra ADN ca lc lp khng cha mt s gene c trng cho nhng sinh vt c nhn tht. iu ny tm thy trong ADN thuc l v ng. Ribosom ca lc lp cng ging

    6

  • ribosom ca sinh vt Procariot: 70S c cu to bi 2 tiu phn 50S v 30S. Ribosom ca E. coli v lc lp thc vt c c tnh min dch ging nhau. Thng qua vic nghin cu sn phm biu hin gene l protein, ngi ta pht hin gia h gene lc lp v h gene nhn trong t bo thc vt c mi quan h cht ch. - Genome ca lc lp khng c kh nng tng hp tt c cc protein c trong chng. - Phn ln polipeptid ca lc lp c tng hp do genome trong nhn t bo. - Nhng polipeptid ny c chuyn vo lc lp theo c ch sau dch m thc hin qua v lc lp. - H thng di truyn lc lp iu khin tng hp cc protein cn cho s pht trin ca chng vi s lng khong 100 polipeptid.

    Bng 1.1. Kch thc mt s ADN lc lp (ct. ADN), ADN nhn v vi khun

    AND Kch thc (bp) Kch thc vng ( m)

    Plasmid 1 200 x 103 - Trc khun E-coli (chromosome) 3,8 x 106 - Trng roi (Euglena) (ct. ADN) 1,4 x 105 44 - 44 Thuc l (Tabaco) (ct. ADN) 1,6 x 105 - Ng (Maize) (ct. ADN) 1,36 x 105 43 u xanh (mungbean) (ct. ADN) 1,21 x 105 39 - Polipeptid c m ho tng hp v ARN trong lc lp m nhim chc nng ca c quan t lin quan n qu trnh quang hp. la ngi ta xc nh c v tr ca cc gene quan trng iu khin qu trnh quang hp nh rbcl, atpBE, pSbA, pstA, psbA. Cc gene ny ng vai tr quan trng trong qu trnh sinh tng hp protein h thng quang ho II. 1.3. H gene ti th (Mitochondria Genome)

    nghin cu h gene ti th iu quan trng u tin l thu c ADN ti th. C th ly v d cy la theo Salech v cng s (1989), phng php phn tch ADN trong ti th gm cc bc sau: - Li tm vi tc chm loi b nhn, lc lp v cc thnh phn khc ca t bo. - Li tm vi tc nhanh tch ti th. - Li tm vi tc chm ly nhn, lc lp v cc thnh phn khc.

    7

  • - X l ADN- ase I phn gii cc cht ADN nhn dnh trong ti th. - Lm sch ti th. - Tch chit mtADN t ti th (mtADN). Nh vy ngi ta c th thu c ADN ca mitochondria tinh sch phc v cho cc cng tc nghin cu tip theo. mtADN th hin mt s khc bit rt ln v mt kch thc v hnh dng. i vi ng vt, mtADN c dng vng v kch thc xp x 15 - 20kb. Ngc li mtADN ca thc vt c nhiu dng (vng.,thng va(cc dng khc), cng nh c kch thc ln hn nhiu: cy bp ci l 200kb, hoc mt loi da l 2500 kb. Do vy genome ti th thc vt tng i ln v gm mt vi phn t ADN. Lng mtADN trong thc vt chim khong 0,5 - 1% lng ADN ca t bo nhng n ng vai tr sng cn cho s pht trin v sinh sn thc vt. Chc nng ca mtADN ging nh ctADN. N c kh nng m hoatng hp mt s lng nh pohlieptid nhng rt quan trng.,phc v cho nhng hot ng ca chnh n. Nhng sn phm chnh m mtADN iu khin tng hp l 3 tiu phn ca cytochromoxydaza, 2 tiu phn ca phc cht cytochrom bc v mt s thnh phn khc (Andre, 1991). Ngoi ra mtADN cn ng vai tr quan trng trong hin tng bt dc c t bo cht (CMS). y l mt c tnh c khai thc trong sn xut cc dng lai nhiu cy trng nh la, ng... Spruill v cng s (1981) ch ra cy ng c s khc nhau gia mtADN trong t bo cy thng v trong t bo cy c hin tng CMS. Khi phn tch sn phm protein th nhng mtADN cy CMS c kh nng tng hp chui peptid 130000 MW, nhng n mt kh nng tng hp chui polipeptid 21000 MW nguyn sinh cht t bo cy bnh thng. Mt khc ngi ta cn thy hin tng bt dc t bo cht cn lin quan n nhng phn t ADN ging nh plasmid tm thy trong ti th. Chng hn ng trong t bo cht ca cy CMS c mang hai phn t AND mch thng vi kch thc 6,2 kb v 5,2 kb. Vic s dng code m ha tng hp protein ca mtADN c nhng im cn lu : trong h thng hot ng m di truyn thng thng code UGA cung cp tn hiu kt thc qu trnh tng hp mt chui polipeptid, nhng ADN ca ti th n li l code ca tryptophan. Ngc li ADN nhn code CGG m ho cho tryptophan th ti th n li m ho arginin. Ngoi ra ngi ta pht hin mt dng mi ca mARN trong genome ti th cy trng v n c gi l ARN - Editing. y l mt...

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