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Cc phng php xc nh s hin din v biu hin ca gen ngoi laiI. Southern blotSouthern blot l mt trong nhng phng php trung tm ca Sinh hc phn t. N cn c tn gi khc l Southern blotting, phng php lai Southern hay phng php lai DNA . Nguyn tc ca Southern blot l mng lai nitrocellulose c kh nng tip nhn DNA c bit t lu v c s dng trong cc nghin cu lai axit nucleic khc nhau vo nhng thp nin 1950 v 1960. Vo thi k ny DNA c nh khng c phn on, ch n gin bao gm DNA tng s c gn trn mng lai nitrocellulose. S ra i ca phng php in di trn gel vo u thp nin 1970 cho php cc on DNA c ct bi enzyme hn ch c th c phn tch da trn c s kch thc ca chng. T bc pht trin tip theo ca phng php l chuyn cc on DNA phn tch t gel ln mng lai nitrocellulose. Phng php ny c E. M. Southern m t ti i hc Edingburgh vo nm 1975. Phng php Southern blot n gin v hiu qu. Mc d c ci tin nhng phng php ang c s dng nhiu phng th nghim sinh hc phn t sai khc khng ng k so vi phng php ban u. Southern blot bao gm cc bc c bn sau: - Ct DNA bng enzyme hn ch thch hp. - in di sn phm ct trn gel agarose. - Lm bin tnh DNA (thng thng khi n cn trn gel): v d c th nhng n vo trong dung dch NaOH 0.5M, DNA si kp s c tch thnh DNA si n. Ch DNA si n mi c th chuyn ln mng lai.
95 - Chuyn DNA bin tnh ln mng lai. Thng thng
mng lai c s dng l mng nitrocellulose. Ngi ta cng c th s dng mng nylon. Mng nitrocellulose in hnh c kh nng tip nhn 100g DNA/cm2, trong khi mng nylon c kh nng tip nhn 500g DNA/cm2. Mt khc mng nylon c kh nng gi DNA chc hn v t t gy hn. Vic chuyn DNA thng c tin hnh bng hot tnh mao dn trong khong vi ting hoc c th dng mt thit b thm chn khng. Nu dng thit b thm chn khng th s nhanh hn, ch mt khong mt ting. Trong qu trnh chuyn, v tr cc on DNA vn c gi nguyn khng thay i. - Lai DNA c c nh trn mng vi mu d (probe) DNA c nh du. Qu trnh ny da trn nguyn tc b sung (gia DNA trn mng lai vi mu d). nh du ngi ta thng s dng P32, biotin/streptavidin hoc mt mu d pht quang sinh hc. - nh v cc phn t lai DNA-mu d. Nu s dng mu d nh du phng x th dng phng php phng x t ghi (autoradiograph) xc nh, nu s dng biotin/streptavidin th dng phng php so mu hoc nu s dng mu d pht quang sinh hc th pht hin bng s pht quang. Phng php Southern blot c thit k xc nh s hin din, kch thc, s lng bn sao, tnh ng dng ca DNA trong mt phc hp. V d, Southern blot c th c s dng pht hin mt gen c bit trong mt genome nguyn vn.
II. Northern blotSau khi E. M. Southern m t phng php Southern blot vo nm 1975, ngi ta dng mt phng php tng t xc nh cc on RNA c bit gi l Northern blot. Phng php ny cn c gi l Northern blotting, phng php lai Northern hay phng php lai RNA. Northern blot bao gm cc bc c bn sau:
96 - RNA (RNA tng s hoc ch mRNA) c phn tch bngin di trn gel agarose.
Hnh 3.1: S m ta phng phap Southern blot
- RNA sau khi phn tch c chuyn ln mng lai (ccphn t RNA gi nguyn v tr nh trn gel).
97 - RNA c nh trn mng c lai vi mu d DNA si n
(hoc RNA) c nh du phng x hoc c gn vi mt enzyme (alkalin phosphatase hoc horseradish peroxidase) to thnh phn t lai RNA-DNA (hoc RNA-RNA) si kp.
Hnh 3.2: S m t phng php Northern blot
- V tr ca mu d c pht hin nh k thut phng x t
ghi nu n c nh du phng x. Trong trng hp mu d c gn vi enzyme th em vi mt c cht khng
98mu. Enzyme lin kt vi n s bin i thnh mt sn phm mu c th nhn thy hoc pht ra nh sng m s c pht hin bng phim X quang mt cch trc tip. Phng php Northern blot cho php pht hin s c mt, xc nh kch thc, trng lng phn t, khi lng mRNA trong cc mu khc nhau. y l mt phng php rt tt phn tch s biu hin ca gen khi chng ta cn nh lng phn bit s khc nhau gia hai mu v n rt nhy bi vy chng ta c th in di mt lng ln RNA tng s hoc mRNA trn gel.
III. Western blotWestern blot l phng php c nhy cao da trn tnh c hiu ca khng th pht hin protein c in di trn gel SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) v chuyn ln mng lai. Western blot cho php xc nh s c mt, trng lng phn t, nh lng protein c mt trong cc mu khc nhau. Western blot cn c tn gi khc l Western blotting hay l phng php lai thm protein. Western blot bao gm cc bc c bn sau: - Protein c phn tch bng in di trn gel SDS-PAGE. - Cc protein c chuyn sang mng lai nitrocellulose, gi nguyn v tr nh phn tch trn gel. - mng lai c nh protein vi mt khng th s cp (primary antibody). Khng th s cp l mt khng th c hiu, s bm vo protein v to thnh mt phc hp protein-khng th i vi protein quan tm. - Tip theo mng lai vi mt khng th th cp (secondary antibody) c enzyme (alkalin phosphatase hoc horseradish peroxidase) i km. Khng th th cp s bm vo khng th s cp ging nh khng th s cp bm vo protein. - Tip tc mng lai trong mt hn hp phn ng c hiu vi enzyme. Nu mi vic u din ra mt cch chnh xc s pht hin thy cc bng bt k ni no c mt phc
99hp protein-khng th s cp- khng th th cp-enzyme hay ni cch khc l bt k ni no c mt protein quan tm. t mt phim nhy cm vi tia X ln mng lai pht hin cc im sng pht ra do enzyme.
-
Hnh 3.3: S m t phng php Western blot
IV. ELISA (Enzymee-Linked Immunosorbent Assay)
100ELISA c m t ln u tin vo nm 1971 v t tr thnh mt phng php c s dng ngy cng rng ri v quan trng hn trong nghin cu, chn on v xt nghim bi v n c kh nng pht hin nhy bn vi mt lng vt cht rt nh. ELISA thay th mt s k thut huyt thanh c in phc tp, cng knh tn nhiu thi gian hn v m rng phm vi phng php pht hin virus cng nh cc marker lin quan n s nhim ca chng. Xt nghim ELISA c th c tin hnh vi mt s phng php nh ELISA trc tip, gin tip, sandwich v cnh tranh. Nguyn tc c bn ca phng php ELISA l khng nguyn ho tan trong dung dch m thch hp c th ph ln b mt plastic (nh polystyrene). Qu trnh ny c th l trc tip hoc thng qua mt khng th. Khi huyt thanh c thm vo, cc khng th c th kt hp vi khng nguyn pha c (solid phase). Xt nghim ELISA c thc hin trong a plastic kch thc 8cm x 12cm, cha 8x12 ging. Mi ging c chiu cao khong 1cm v ng knh l 0,7cm (Hnh 3.4).
Hnh 3.4: a plastic s dng tin hnh xt nghim ELISA
10 1
Cc khng th s dng trong phng php ELISA c gn vi enzyme bng lin kt ng ho tr. Khng nguyn c gn vi ging plastic v khng th lin kt vi enzyme c gn vi khng nguyn. Khng th khng gn khng nguyn s b ra tri i. Enzyme c gi li v v vy lng khng th gn enzyme c pht hin bng cch cho thm vo mt c cht lm thay i mu do hot tnh ca enzyme. mu to thnh l t l vi lng enzyme bm ging plastic, t suy ra lng khng th, sau tip tc suy ra lng khng nguyn (Hnh 3.5). Tnh nhy ca ELISA l do s khuych i bi hot tnh enzyme. Mi mt phn t enzyme bm vo khng th c th to ra hng ngn phn t mu do hot tnh enzyme. Trc khi cc khng th gn enzyme c th c s dng rng ri, cc khng th phng x c s dng trong k thut min dch phng x (radio immuno assays-RIA). K thut RIA nh l mt t ph c ngha v ngi sng to ra n l Rosalyn Yalow c nhn gii thng Nobel Sinh l v Y hc vo nm 1977.
Hnh 3.5: Nguyn tc ca phng php ELISA
102
V. Phng php PCRPhng php PCR (polymerase chain reaction-phn ng tng hp dy chuyn nh polymease) l mt trong nhng phng php c s dng rng ri nht trong lnh vc Sinh hc phn t. Phng php ny do Kary Mullis pht minh vo nm 1985 v c gii thiu ln u tin ti Hi tho ln th 51 Cold Spring Harbor vo nm 1986 v ng nhn c gii thng Nobel Ho sinh hc vo nm 1993. Phng php PCR cho php tng hp rt nhanh v chnh xc tng on DNA ring bit. y thc s l phng php hin i v thun tin cho vic xc nh s c mt ca mt gen no trong t bo vi chnh xc cao. Phng php ny da trn s khm ph hot tnh sinh hc nhit cao ca DNA polymerase c tm thy trong cc sinh vt a nhit (vi khun sng trong cc sui nc nng). Phn ln cc DNA polymerase ch lm vic nhit thp. Nhng nhit thp, DNA xon cht v vy DNA polymerase khng c nhiu kh nng lm bin tnh phn ln cc phn ca phn t. Nhng cc polymerase chu nhit ny hot ng nhit rt cao, c th ln n 100oC. nhit ny DNA (dng thng) s b bin tnh. 1. Cc thnh phn ch yu ca phn ng PCR 2.1. DNA mu (DNA template) y l mu DNA sinh hc m chng ta mun khuych i. Phn ng PCR ti u xy ra trn DNA tht tinh sch nhng phn ng PCR vn cho kt qu tt vi DNA thu nhn trc tip t dch chit t bo. Lng mu DNA s dng c khuynh hng gim khi s dng cc enzyme DNA polymerase cho hiu qu cao (