94 Chng 3
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 (
