1

KY THUAT DI TRUYEN
2
QUA 1kNH NCH|EN CUU CENE
C0 LAP
CENE
1och ch|et
DNA, kNA
Phon t|ch
d|nh t|nh,
d|nh |ucng
1oc
dcng
PCk,
k1-PCk
1HU NHAN
CENE
C|o|
tr|nh tu
Coc phucng
phop |n s|||cc
XAC [NH CAU
1kUC CENE
1M H|EU CHUC
NANC CUA CENE
Ncrthern
b|ct
5cuthern
b|ct
Coy toc
dct b|en
Ph|en mo
d|ch mo
|n v|trc
...
UNC DUNC
1cng hcp
nhon toc
Muc t|eu : H|eu rc cou truc vo chuc nong cuo gene
de ung dung voc coc ||nh vuc khco hcc vo dc| scng
|en
d|
c
0D
5u dung coc
enzyme, vectcr
3
N0| DUNC
. 1hu hc| nuc|e|c oc|d
2. Phon t|ch d|nh t|nh vo d|nh |ucng nuc|e|c oc|d
3. 5u dung coc enzyme thcng dung trcng s|nh hcc phon tu
4. Lo| phon tu
5. 1oc dcng
6. PCk
7. C|o| tr|nh tu nuc|e|c oc|d
8. 8|en dc| vot ||eu d| truyen vo chuyen vot ||eu d| truyen do
b|en dc| voc te boc
4
1HU H0| NUCLE|C AC|D
. PHU0NC PHAP 1ACH CH|E1
- 1och ch|et DNA
- 1och ch|et kNA
2. PHU0NC PHAP L 1AM
- Ly tom phon dcon
- Ly tom dong ty trcng 1hu nhon nuc|e|c oc|d t|nh soch
3. PHU0NC PHAP 5AC K
1hu mkNA
5
1ACH CH|E1 DNA & kNA
DNA kNA
1h|ccyonote
. Pho mong te boc, mong nhon Chot toy (5D5[ guon|d|n|um
+ s|eu om, .. L|C|, Ureo, ..
+ cc hcc, ..
2. Lco| prcte|n Phenc| pH8 : Phenc| pH4 :
Ch|crcfcrm:|AA Ch|crcfcrm:|AA
Khoc Khoc
3. 1hu hc| nuc|e|c oc|d :
1uo Ethonc| tuyet dc| Ethonc| tuyet dc|
|scprcponc|, .. |scprcponc|, ..
8ccm Hop phu tren s|||co Hop phu tren s|||co
Lcc 1hu tren mong |cc 1hu tren mong |cc
6
PHA MANC 5|NH H0C 8ANC CHA1 1A
Chot toy |cn hco cc toc dung
pho mong monh, khcng |cn
hco cc toc dung pho mong
nhe hcn.
Chot toy, |o phon tu |ucng
cuc, se ket hcp vc| prcte|n
mong vo coc phon tu
phcsphc||p|d |om pho vc cou
truc mong
7
L 1AM PHAN 0AN
Ly tom phon dcon (o[
vo phon dcon vung (b[
phon toch coc phon tu
trcng hcn hcp duo tren
khc| |ucng cuo chung.
1hc| g|on vo |uc |y tom
ducc xoc d|nh chc tung
nhcm phon tu.
Dung d|ch |y tom cc ty
trcng thop hcn coc
phon tu con phon toch
8
L 1AM ANC 1 1k0NC
Ly tom dong ty trcng
(|scpycn|c[ phon toch
coc phon tu trcng hcn
hcp duo tren ty trcng
cuo chung.
1hc| g|on vo |uc |y tom
|o chung chc coc nhcm
phon tu.
Dung d|ch |y tom cc ty
trcng vc| g|o tr| d| tu
thop hcn den coc hcn
ty trcng cuo coc phon
tu con phon toch
9
1HU H0| NUCLE|C AC|D, PHACE 5AU L 1AM
ANC 1 1k0NC
10
CAC PHU0NC PHAP 5AC K
5oc ky
|cc ge|
5oc ky
troc
dc| |cn
5oc ky
o| |uc
K1-KN
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1HU H0| mkNA 8ANC 5AC K A| LUC
12
CAU H0| PHAN
. Nhung khoc b|et trcng phucng phop toch ch|et-t|nh soch DNA vo kNA, v| soc
|o| cc nhung khoc b|et dc 7
2. Phucng phop tuo, 8ccm cc nhung uu nhucc d|em g| 7
3. Khoc b|et g|uo |y tom phon dcon vo |y tom dong ty trcng 7 Ung dung cuo
tung |co| |y tom 7
4. Phucng phop toch ch|et-t|nh soch DNA p|osm|d cc g| khoc vc| toch ch|et-t|nh
soch DNA bc gene 7
13
PHAN 1CH [NH 1NH VA [NH LU0NC
NUCLE|C AC|D
. |EN D|
- Ce| ogorcse
- Ce| pc|yocry|om|de
2. QUANC PH0 KE : c mot dc quong (0D 0pt|co|
dens|ty[
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|EN D| DNA 1kEN CEL ACAk05E
- |en d| thec phucng nom ngong
- C|o the |o ge| ogorcse
- Phot h|en vc| mou nhucm
eth|d|um brcm|de, ... phot huynh
quong duc| den tu ngco|
- Kho nong phon g|o| trung b|nh,
phu thucc voc nh|eu yeu tc, doc
b|et |o ncng dc ogorcse
15
|EN D| 1k0NC 1kU0NC XUNC (PUL5ED-F|ELD
ELEC1k0PH0kE5|5[
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|EN D| 1kEN CEL P0LACkLAM|DE
- |en d| thec phucng thong dung
- C|o the |o ge| pc|yocry|om|de
- Phot h|en vc| mou nhucm
Cccmoss|e b|ue, nhucm boc, phot
h|en tren ph|m t|n h|eu dcng v|
phcng xo
- Kho nong phon g|o| coc, phon
b|et ducc nhung tr|nh tu ch| coch
nhou nuc|ect|de
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|EN D| Pk01E|N 1kEN CEL
P0LACkLAM|DE
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|EN D| 2 CH|EU Pk01E|N 1kEN CEL
P0LACkLAM|DE
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K 1HUA1 kFLP (kE51k|C1|0N FkACMEN1
LENC1H P0LM0kPH|5M A HNH KCH
1HU0C CAC 1kNH 1U CA1 C|0| HAN
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VN1k (VAk|A8LE NUM8Ek 0F
1ANDEM kEPEA15[
Phon t|ch VN1k de nhon b|et t|nh do h|nh d| truyen c co
the. 5u dung mou dc ket hcp vc| kE de phon t|ch VN1k
21
UNC DUNC K
1HUA1 |EN D|
1k0NC NCH|EN CUU
PHA HE
V| du ve v|ec su dung PCk vo d|en
d| trcng xoc d|nh pho he.
oy |o pho he cuo 5o hcong, vc, 3
ccn, boc s| g|o d|nh vo coc nguc|
hou
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K 1HUA1 ELEC1k0PH0kE1|C M08|L|1 5H|F1
A55A : PHA1 H|EN NHAN 10 1kAN5
Coc phon dcon prcte|n ducc u vc| mct dcon DNA do ducc donh dou phcng xo vo
cc mong mct tr|nh tu C|5. Coc tc hcp tren sou dc se ducc phon t|ch bong d|en d|
khcng b|en t|nh (dc| vc| prcte|n[. Coc phon dcon DNA khcng gon prcte|n se d|
chuyen xo hcn coc phon dcon trcng dc tr|nh tu C|5 ||en ket vc| prcte|n (7, 8 trcng
h|nh[. Nhu voy coc phon dcon prcte|n tucng ung vc| coc tc hcp 7 vo 8 se ducc xoc
d|nh.
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CAU H0| - PHAN 2
. Coc d|em khoc b|et trcng phucng phop t|en honh d|en d| tren ge| ogorcse vo ge|
pc|yocry|om|de
2. Coc ung dung cuo d|en d| tren ge| ogorcse, d|en d| tren ge| pc|yocry|om|de
3. Phucng phop dc mot dc quong (0D[ :
- Nguyen |y vo coch xoc d|nh hom |ucng nuc|e|c oc|d
- Nguyen |y vo coch xoc d|nh dc soch cuo nuc|e|c oc|d
4. Nguyen |y vo coch t|en honh phucng phop kFLP (kestr|ct|cn Frogment Length
Pc|ymcrph|sm[
5. Nguyen |y vo coch t|en honh phucng phop VN1k (Vor|ob|e Number cf 1ondem
kepeots[
6. Nguyen |y vo coch t|en honh phucng phop E|ectrcphcret|c Mcb|||ty 5h|ft Assoy
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5U DUNC CAC ENZME 1H0NC
DUNC 1k0NC 5|NH H0C PHAN 1U
. ENZME C|0| HAN (kE51k|C1|0N ENZME[
2. P0LMEkA5E (DNA, kNA[
3. NUCLEA5E (DNose, kNose[
4. L|CA5E
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M01 50 ENZME C|0| HAN (kE[
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M01 50 ENZME C|0| HAN (kE[
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CAC END0- & EX0NUCLEA5E
Lco| enzyme Cc chot V| tr| nhon b|et vo cot tren DNA, kNA
28
LAP 8AN 0 CA1 C|0|
HAN (kE51k|C1|0N MAP[
1r|nh tu DNA con ngh|en cuu ducc cot
vc| nh|eu kE, thec coc tc hcp cot dcn,
cot phc| hcp. Coc son phom cot sou dc
ducc phon t|ch bong d|en d| vo xu |y vc|
coc phon mem v| t|nh de xoy dung bon
dc cot g|c| hon.
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K 1HUA1 DNA5E |
F001Pk|N1|NC : XAC [NH
1kNH 1U C|5 1kEN DNA
Mct dcon DNA ducc donh dou phcng xo
c mct dou 5 vo ducc u chung vc| coc
phon dcon prcte|n ducc xem |o cc tucng
toc vc| DNA noy. 5ou dc, tc hcp ducc
mong phon cot bong enzyme Dnose |.
Nc| noc tren DNA cc gon prcte|n, nc| dc
se ducc boc ve khc| su phon cot noy, vo
h|nh thonh dou chon (g|eng -2 vo 0
tren h|nh[. c ch|nh |o tr|nh tu C|5. K|ch
thucc cuo tr|nh tu cc the ducc nhon b|et
kh| sc sonh vc| thong DNA (M tren h|nh[
30
CAU H0| PHAN 3
. Coch xoy dung bon dc cot hon che (restr|ct|cn mop[
2. Coch t|en honh phucng phop DNose Fcctpr|nt|ng, ung dung cuo phucng phop
3. Ung dung cuo coc nuc|eose : Nuc|eose 5, DNose |, kNose A, kNose H,
4. Ung dung cuo coc pc|ymerose : reverse tronscr|ptose, DNA pc|ymerose, 5P6 kNA
pc|ymerose, 1oq pc|ymerose, DNA pc|ymerose |, 1erm|no| tronsferose
5. Ung dung cuo 14 pc|ynuc|ect|de k|nose, cuo o|ko||ne phcsphotose
LA PHAN T
1. OANH OA MA OO ONA, RNA,
OLGONCLKOTOK
2. CAC KK LA
W Lu trong phu long
W Lu tren phu run (Southern, Northern,
dot blot)
W Lu tu cho
3. NG ONG
CAC PHAN T ONG OANH OA HOA HOC
Co aI Iuc voI anfI-
IgoxIgonIn
ung phoI hop voI
dATP, dCTP,
dCTP, dTTP
Co aI Iuc voI
sfropfavIdIn
ung phoI hop voI
dATP, dCTP,
dCTP, dTTP
OANH OA BANG BOTN
anh dau mau do |probo) IaI voI frnh fu muc fIou Phaf hIon phan fu IaI
fhong qua phuc hop avIdIn/chaf phaf huynh quang
PHONG PHAP NCK TRANSLATON
W uc Io fron 2 mach cua
NA bang naso I
W NA poIymoraso I su
dung haof fnh 5-3
oxonucIoaso gam mach
NA fhoo chIou 5-3
W NA poIymoraso I vua
fhuy phan vua fong hop
mach moI bu vao Io frong
voI cac dNTP |dATP, dCTP,
dCTP, dTTP, d!TP danh
dau)
W Kof qua Ia probo duoc
danh dau
PHONG PHAP RANOOM PRMNG
W Ben tInh much do ONA
W But cup to hop mo nguu
nhen tren 2 much
6
4
= 1296 prmers
W ONA polymeruse tong hop bu
vuo cho trong bung cuc dNTP
trong do co nucleotde dunh
duu
W Ket quu lu probe dunh duu
PHONG PHAP
OANH OA
OLGONCLKOTOK
PHAN NG
PHOSPHATASK
PHAN NG
KNASK
PHONG PHAP OANH OA
OLGONCLKOTOK S ONG
TKRMNAL TRANSFKRASK
TONG HOP MA OO
RNA BANG PHKN
MA N VTRO
W Trnh fu so dung Iam probo duoc fao
dong vao vocfor bIou hIon
W Tuy mach muon dung Iam probo so
su dung promofor fuong ung
W RNA poIymoraso nhan bIof promofor
dac hIou, gan vao va fong hop RNA
W NucIoofIdo fu do dung frong qua
frnh phIon ma co mang mof phan
nucIoofIdo danh dau
W Kof qua Ia mau do RNA danh dau
|rIboprobo)
TONG HOP MA OO RNA BANG PCR VA
PHKN MA N VTRO
gene A
T? promoter
PCR
Phen mu in rilro vo T?
RNA polymeruse
Muu do RNA
Thuy vI tuo dong thI su dung PCR de gun them trInh tu promoter phu
hop
Quu trInh tep theo gong nhu tuo dong
SOTHKRN BLOT
SOTHKRN BLOT (tep)
Tuch chet & cut
ONA bung enzyme
go hun
Oen d Chuyen len mung lu Lu vu phut hen phun tu lu
Ben tInh ONA Ten lu
LA TRKN PHA RAN
& PHAT HKN PHAN
T LA BANG OONG
V| PHONG XA
Co d|nh
ONA len
mung lu
Lu vo muu
do dunh duu
phong xu
Ruu mung lu
Thuc hen
phong xu tu gh
SOTHKRN -
NORTHKRN
BLOT
KKT QA NORTHKRN BLOT
ChI chu : !N : Mau chung khon cam ung, 48h : mau cam ung fao E-gIobIn sau 48
gIo, 96h : mau cam ung fao E-gIobIn sau 96 gIo. Mau do dong vj phong xa
OOT BLOT
Tuch chet ONA, RNA Ben tInh
(neu co) Out len mung lu Ten
lu Lu vo muu do (trInh tu du
bet) Phut hen phun tu lu
Oung de phut hen, houc d|nh luong
tuong do kh so vo thung hum
luong du bet
Phuong phup cu ben lu reverse
dot blot. TrInh tu du bet duoc co
d|nh tren gu the con trInh tu cun
bet (muc teu) duoc dunh duu
WKSTKRN BLOT
Oen d tren
gel
polyucrylumde
Chuyen len
mung lu
Len ket vo khung the 1 &
2. Ruu de lou khung the
thuu
Phut hen muu
thong quu phun ung
enzyme
CAT MA CHO LA TA CHO (N STU
HYBROZATON)
KKT QA LA TRKN NHKM SAC THK
LA TA CHO TRKN PHO
Co d|nh muu (Iormol, dong lunh) Cut lut mong (mcrotome) Co d|nh
tren lum kInh Xu ly muu truoc kh lu Ten lu Lu vo muu do
dunh duu hou hoc huy dong v| phong xu Ruu Phut hen phun tu lu
Quun sut duo kInh hen v
NGYKN TAC CA
MCROARRAY
- Tuch mRNA tu te buo nuo cuy
- Phen mu nguoc thunh cONA co dunh
duu huynh quung khuc nhuu
- Tron chung 2 lou cONA dunh duu
- Ruu, lou cuc thunh phun thuu
- Oo cuong do phut huynh quung o tung
dem :
Oem phut muu vung : gene beu hen
nhu nhuu trong 2 lou te buo
Oem phut muu xunh : gene cuu te buo
nuo trong glucose beu hen munh hon
Oem phut muu do : gene cuu te buo
nuo trong ethunol beu hen munh hon
KKT QA MCROARRAY
V O : PHAT HKN
BKNH HONG CA
HNH LKM BANG
SOTHKRN BLOT
- Tuch ONA
- Cut bung enzyme Mst
- Chuy den d
- Lu vo muu do E-globn dunh duu
phong xu
- Phut hen bung phong xu tu gh
muu ONA benh duu vuo kIch thuoc
trInh tu cho tIn heu lu
KY THAT O BO TRKN NHKM SAC THK
(CHROMOSOMK WALKNG)
KKT QA RFLP TRONG NGHKN C
PHA HK
CAU H0| PHAN 4
. Coch donh dou mou dc bong dcng v| phcng xo 7 8ong hco hcc 7 Uu nhucc
d|em cuo tung |co| toc nhon 7
2. Coch donh dou mou dc cc bon chot |o DNA, kNA, c||gcnuc|ect|de
3. 5cuthern b|ct z Ncrthern b|ct z dot blot ducc thuc h|en nhu the noc 7
4. Chc v| du ve v|ec su dung reverse dct b|ct
5. Coch t|en honh 5cuthern b|ct, Ncrthern b|ct, dct b|ct
6. Ung dung cuo 5cuthern b|ct, Ncrthern b|ct, dct b|ct
7. Coch t|en honh vo ung dung cuo phucng phop |o| to| chc
8. Coch phot h|en phon tu |o| vc| mou dc dcng v| phcng xo 7 Hco hcc 7
. Coch t|en honh phucng phop su dung m|crcorroy vo ung dung cuo m|crcorroy
0. Ky thuot d| bc tren nh|em soc the : coch |om vo ung dung
TAO OONG
HA CHKN LOC TAO OONG
N VVO & N VTRO
NGYKN TAC TAO OONG (N VVO)
CAC BOC TAO OONG
Chuan bj
vocfor
Chuan bj
frnh fu
fao dong
|Insorf)
Tao vocfor
faI fo hop
Ion nap
vocfor faI fo
hop vao vI
khuan
Chon Ioc
dong can
fm
Xu Iy
enzyme
luo
Juu so
Ie, Juu
bung
Noi
reclor/
inserl
W Hou
bien nup
W Dien
bien nup
W Bien
nup bung
sieu um,
W DNA :
luch chiel
DNA bo
gene, PCR,
long hop
W RNA :
luch chiel
RNA ru
RT, RT-
PCR
W IaI voI
mau do
dac hIou
W PCR
voI moI
dac hIou
W Su
dung
khang
fho dac
hIou
CHAN B|
cONA OK TAO
OONG
mRNA cONA
gun them cuc
lnker co mung
trInh tu nhun bet
cuu RK
Cut bung RK
tuong ung tuo duu
so le
X LY OOAN GAN CHKN CO OA BANG
W Can fhom cac
IInkors mang
frnh fu nhan bIof
cua R
W Vocfor co chua
cac frnh fu nhan
bIof cua R fuong
ung
TAO OONG BANG
PCR
W MoI |prImor) dung frong phan
ung PCR co mang frnh fu nhan bIof
cua R
W San pham PCR duoc caf bang R
phu hop fao dau so Io
W NoI voI vocfor da xu Iy voI cung
IoaI R
TAO OONG TRONG
PLASMO
- Tuo plusmd tu to hop
- Ben nup te buo E. coIi : u chung vo
plusmd TTH co hen den CuCl2 ; tuo
soc nhet
- Nuo tren dIu thuch co umpclln de
chon dong v khuun co thu nhun
plusmd TTH
- Thu nhun cuc dong te buo co chuu
plumd TTH
pBR322
W Plusmd the he thu nhut
W Plusmd the he thu hu :
pR322
W Plusmd the he thu bu :
p!C, pSP, IuoscrIpf, ..
PLASMO pC18/19
TAO OONG TRONG
PLASMO pC
BO GKNK PHAGK VA S T RAP
TAO OONG VO VKCTOR LA PHAGK
Thu nhun ONA phuge
Lou bo trInh tu khong
cun cho su dong go
Gun trInh tu cun tuo dong
vuo thuy cho trInh tu lou bo
Oong go vuo vo boc cuu
phuge
Oem xum nhem te buo v
khuun de chuyen trInh tu
cun tuo dong vuo v khuun
TAO OONG cONA
TRONG PHAGK O
Oem dung d|ch phuge tu to hop tru tren lop
te buo v khuun phu duy mut thuch tuo dIu
ly gu
TAO OA LY GA (PLAQK ASSAY)
Be mut thuch phu mot lop te buo E. coIi Them huyen phu vrus tu to
hop Phu tep len tren mot lop ugur mem Mo dIu ly gu tuong
ung vo vung ly gu do mot phun tu vrus guy ru
PHAT HKN OA LY GA CO CHA OONG CAN TM
Hop thuch vo cuc dIu ly gu

Ap mung lu len mut thuch

mung lu trong dung d|ch kem de

ly gu phuge gu phong ONA phuge

Ten lu - Lu vo muu do

Phut hen phun tu lu

TONG HOP M13 MACH OO
COSMO VKCTOR PHC?9
TAO OONG BANG VKCTOR COSMO
TAO NGAN HANG BO GKNK
TAO OONG VO BAC (NHKM SAC
THK NHAN TAO CA V KHAN
TAO OONG TRONG
VKCTOR BK HKN
W Vector beu hen lu vector co
mung cuc promoter cho phep phen
mu trInh tu gun chen (nsert)
W Su dung RNA polymeruse phu
hop vo promoter de phen mu
much muon su dung lum khuon
PHKN MA & O|CH MA TRNH T TAO OONG
TAO OONG BANG VKCTOR CON THO
Muc teu : Nhun bun vu chon loc trInh tu gun chen (nsert)
trong 2 lou te buo chu khuc nhuu
PHONG PHAP N
ung do fao mang IaI dung
phaf hIon dong can fm
ung do chon Ioc dong can fm
fron nhIou IoaI moI fruong
khac nhau
CHON OONG TA TO HOP
Chon cuc dong co
mung vector tu to hop
duu tren duc tInh
khung 2 khung snh
cuu vector pBR322
CHON OONG TA TO HOP
Chon dong faI
fo hop dua
fron su khang
1 hay 2 khang
sInh cua vI
khuan sau
bIon
nap|ampIcIIII
n va
fofracycIIn)
Chon dong
faI fo hop
dua fron
mau khuan
Iac
|xanh/frang)
LA TRKN KHAN LAC -
CHON OONG CAN TM
BANG LA PHAN T
Muu do duoc suy ru tu trInh tu umno ucd duu
theo mu d truyen, duoc tong hop vu dunh duu,
suu do dem lu vo mung lu co mung cu trInh
tu ONA du duoc co d|nh
CHON OONG CAN TM
BANG KHANG THK
W Tao mof ngan hang bIou
hIon.
W Cam ung cho cac profoIn
bIou hIon
W Phaf hIon dong can fm
frong ngan hang bIou hIon
voI khang fho danh dau va
dac hIou cho profoIn bIou
hIon
CA HO PHAN 5
1. Vocfor dung do fao dong can co nhung dac dIom g
2. Co nhung IoaI vocfor nao 7 !u nhuoc dIom cua cac IoaI ay 7 !ng dung cua
chung 7
3. ac dIom cua cac vocfor pIasmId p!C, pSP, IuoscrIpf
4. Mo fa cach su dung vocfor Ia phago
5. Vocfor bIou hIon dung do Iam g 7 Cho v du
6. Vcofor con fhoI dung do Iam g 7 Cho v du
7. Mo fa cach fao ngan hang bo gono
8. Mo fa cach fao ngan hang cNA
9. Cach phaf hIon dong can fm bang khang fho
10. Cach phaf hIon dong can fm bang mau do nucIoIc acId
11. Cach bIon nap vocfor faI fo hop vao fo bao vI khuan
PCR (POLYMKRASK CHAN RKACTON)
CAC 1HANH PHAN CUA PHAN UNC PCk
. Pr|mer (mc|[
Mc| pho| thco mct sc d|eu k|en cc bon : ([ do| khcong 8-24 bose, (2[
1m cuo 2 pr|mer gon nhou, (3[ [C:C} 40-60X, (4[ khcng h|nh thonh
pr|mer d|mer, (5[ khcng pho| |o tr|nh tu |op |o|.
2. DNA bon mou (temp|ote[
Hom |ucng du nhung khcng quo coc, t|nh soch khcng chuo coc chot
uc che phon ung (5D5, chot uc che tu mou (hemcg|cb|n, soc tc, ..[,
hepor|n, ..[
3. Ncng dc MgC|
2
Con chc hcot dcng cuo 1oq pc|ymerose , hom |ucng quo coc se toc
nh|eu son phom ky s|nh, quo thop th| |om g|om h|eu quo nhon bon
cuo enzyme
4. dN1P
1honh phon 4 |co| dN1P pho| con bong , hom |ucng thucng khcng dc|
nhung cc the thoy dc| tuy d|eu k|en thuc te
5. Enzyme
ucc chcn tuy muc d|ch su dung :
W 1oq DNA pc|ymerose (1hermus oquot|cus[ : thcng dung nhot, hcot t|nh
pc|ymerose kho monh (35-00 nu/g|oy[, cc hcot t|nh 53 excnuc|eose
5tcffe| DNA pc|ymerose : t|nh ch|u nh|et coc hcn, khcng cc hcot t|nh 5-3
excnuc|eose, hcot dcng ducc c phc MgC|2 rcng mu|t|p|ex PCk
1oq vo 5tcffe| DNA pc|ymerose them 3dA voc son phom PCk
W Vent/DeepVent DNA pc|ymerose (1hermcccccus ||tcro||s[ : t|nh ch|u nh|et
rot coc, nhon bon ducc nhung dcon DNA rot do|, cc hcot t|nh 3-5
excnuc|eose t|nh trung thuc coc hcn 1oq pc| 5-5 |on, toc son phom dou
bong
W Pfu DNA pc|ymerose (Pyrcccccus fur|csus[ : cc co 2 hcot t|nh 5-3 vo
3-5 excnuc|eose, t|nh trung thuc coc hcn 1oq pc| 2 |on, thucng dung trcng
cyc|e sequenc|ng
W 1th DNA pc|ymerose (1hermus thermcph||us[ : vuo cc chuc nong
pc|ymerose vuo cc chuc nong ph|en mo ngucc (k1[ dung trcng phon ung
k1-PCk
W U|1mo DNA pc|ymerose (1hermctcgo mor|t|mo[ : t|nh ch|u nh|et rot coc,
cc hcot t|nh 3-5 excnuc|eose t|nh trung thuc rot coc
CAC 1HANH PHAN CUA PHAN UNC PCk (t|ep[
5. Chucng tr|nh PCk :
W Nh|et dc : b|en t|nh 4-5qC , |o| < 1m cuo coc pr|mer ~ 5qC , kec
do| - 72qC
W 1hc| g|on cuo tung chu ky tuy thucc th|et b| vo k|ch thucc son phom
W 5c |ucng chu ky thucng 40
V| du : chu ky - 5qC 5
35 chu ky 4qC 30
55qC 30
72qC 45
chu ky 72qC 0
6. 1h|et b| : Nhung co| t|en moy |uon nh|et ||en quon den ([ m|crct|ter p|ote
6 g|eng, (2[ coc b|cck nh|et r|eng b|et trcng moy, (3[ b|cck dung chc |om
k|nh de t|en honh |n s|tu PCk.
7. Dung cu : 0ng Eppendcrf thonh mcng (th|n-wo||[, dou t|p cc ph|n |cc
CAC 1HANH PHAN CUA PHAN UNC PCk (t|ep[
NHUNC HAN CHE CUA K 1HUA1 PCk
CACH KHAC PHUC
. Ngco| nh|em coc dc kho nong nhon bon DNA monh
Phon ch|o khu vuc th| ngh|em, su dung dou t|p cc ph|n |cc, gong toy
thoy thucng xuyen, ch|eu t|o UV khu vuc th| ngh|em, su dung he thcng
dU1P-ung,
2. 5o| sct cuo DNA pc|ymerose trcng quo tr|nh tcng hcp
Lop |o| phon ung PCk neu con th|et, su dung |co| pc|ymerose cc t|nh
trung thuc coc nhu Pfu, DeepVent, U|1mo pc|ymerose.
3. Ket quo phu thucc rot |cn voc tcon bc thoc toc
Ky thuot v|en con ducc doc toc tct, tu dcng hco, su dung hco chot pho
son (k|t[ do ducc chuon hco
4. 1h|et b| dot t|en
M01 50 PHU0NC PHAP PCk
. PCk : su dung cop mc| , son phom PCk ducc phot h|en bong d|en d|
2. Nested / 5em|-nested PCk : su dung cop mc| ngco| vo mc|/ cop
mc| trcng de tong t|nh doc h|eu vo dc nhoy cuo phon ung PCk
3. Mu|t|p|ex PCk : su dung nh|eu hcn cop mc| trcng phon ung PCk de
phot h|en dcng thc| nh|eu tr|nh tu DNA cuo toc nhon (A[ hoy nh|eu toc
nhon (8[
(A[
(8[
M01 50 PHU0NC PHAP PCk (t|ep[
4. AP-PCk (Arb|trory Pr|med[/kAPD (kondcm Amp||f|ed Pc|ymcrph|c
DNA[ : su dung tc hcp coc pr|mer ngou nh|en de xoc d|nh dou on d|
truyen cuo |co|
([
(2[
5. kACE (kop|d Amp||f|cot|cn cf cDNA Ends[ : nhon bon ho| dou mut 5
vo 3 cuo tr|nh tu excn vc| mc| doc h|eu vo mc| chung
6. |n s|tu PCk : thuc h|en phon ung PCk ngoy tren mc/te boc
7. PCk d|nh |ucng : d|nh |ucng son phom PCk thec thc| g|on thuc duo
tren mou dc phot huynh quong
AAAAAAAA
111111
AAAAA
1111111
k1-PCk
Gom 2 buoc :
1. RT (Phen mu
nguoc) : tuo cONA
2. PCR : nhun bun
cONA
Oung de nhun bun
RNA
kEAL-1|ME PCk
Dung d|nh |ucng DNA hcoc kNA (reo|-t|me k1-PCk[
|nh |ucng duo tren sc t|n h|eu huynh quong/phon ung mo moy dcc ducc
1oc nhon donh dou thucc ho| nhcm :
W Chot phot huynh quong ||en ket vc| DNA moch dc| (58k Creen, Eth|d|um
brcm|de[ se phot huynh quong
W Chot phot huynh quong dung donh dou mou dc doc h|eu
1oqMon prcbe
KE1 QUA PCk kEAL-1|ME
C
A
8
10
1
ban sao
10
1
ban sao
Dung ducng chuon (stondord
curve[ vc| mct chot chuon cc
ncng dc do b|et.
Hom |ucng cuo thonh phon con
phot h|en ducc t|nh duo tren
ducng chuon noy
Ct (Cyc|e threshc|d - chu ky ngucng[ :
chu ky PCk c dc t|n h|eu doc h|eu vuct
khc| t|n h|eu nen
Ch| chu : ducng mou dc |o tn h|eu nen
1A0 D0NC 5AN PHAM PCk
1oq pc|ymerose thucng them A
voc dou 3 su dung 1-vectcr
de toc dcng son phom PCk
Ccn gc| |o 1-A c|cn|ng
PCk NCU0C
Dung nhon bon vung nom
ho| ben mct vung do b|et
tr|nh tu
PCk 1A| 10 H0P
Dung de nc| nh|eu tr|nh tu
ngon thonh mct tr|nh tu do|, V|
du : nc| prcmcter cuo gene A
vc| vung mo hco cuo gene 8
C|A| 1kNH 1U NUCLE01|DE (5EQUENC|NC[
PHU0NC PHAP MAXAM-C|L8Ek1
(H0A H0C[
Nguyen |y : duo tren su phon cot
hco hcc DNA
Coc bucc ch|nh :
. 1hu nhon tr|nh tu DNA con g|o|
2. 8|en dc| hco hcc doc h|eu coc
bose
3. Lco| bc coc bose b|en dc|
4. Cot moch DNA c v| tr| b| |co|
bc bose
PHU0NC PHAP 5ANCEk
(ENZME H0C[ (D|DE0X
5EQUENC|NC ME1H0D[
Nguyen |y : duo tren su tcng hcp
DNA ket hcp vc| tung |co|
d|decxynuc|ect|de
Coc bucc ch|nh :
. 1hu nhon tr|nh tu DNA con g|o|
2. Nhon bon tr|nh tu noy vc|
dN1P vo tung |co| ddN1P
|en d| phon toch coc tr|nh tu DNA toc thonh cuo ho| phon ung

cc ket quo : tu dcng hcoc quo phcng xo tu gh|

PHU0NC PHAP C|A| 1kNH 1U MAXAM-C|L8Ek1
Coc phon ung g|o| tr|nh tu :
C : Hydroz|ne trcng NoC| cot
bose C
C : C ducc methy| hco bong
d|methy|su|fote sou dc b| cot
bong k|em
C + A : Fcrm|c oc|d cot ||en ket
g|uo bose vo ducng
1 + C : Hydroz|ne cot vcng
pyr|m|d|ne
8ucc cuc| :
P|per|d|ne cot coc bose b|en dc|
KE1 QUA C|A| 1kNH 1U H0A H0C
Ket quo cuo ky thuot phcng
xo tu gh| (outcrod|cgrophy[
PHU0NC PHAP
C|A| 1kNH 1U
5ANCEk
C|A| 1kNH 1U 1U 0NC
M01 50 8|EN PHAP CA 01 8|EN |EM [NH HU0NC
The Single-Priming Method
PHU0NC PHAP 5U
DUNC 2 Pk|MEk
01 8|EN
PHU0NC PHAP KUNKEL
dut : dU1Pose
ung : uroc||-DNA-g|yccsy|ose
(W.P. 5temmer (4[ Prcc. Not|. Acod. 5c|. U5A : 0747-075[
PHU0NC PHAP 1A| 5AP XEP DNA (DNA 5HUFFL|NC[
Ccm coc bucc :
uc |c DNA bong Dnose |
1|en honh PCk khcng cc mc| : - 8|en t|nh
- 1o| bot cop
- Kec do|
1oc ro nhung tr|nh tu DNA |o|
CHUEN VA1 L|EU D| 1kUEN 0 1HUC VA1
Su dung v khuun Agrobuclerium
lumejuciens co mung plusmd T
tu to hop de chuyen gene vuo te
buo thuc vut
M01 50 8|EN PHAP CHUEN VA1 L|EU D|
1kUEN
Phuong phup
bun gene
Phuong phup
ben nup
Phuong phup
v tem
Phuong phup
dung vector
vrus
CHUEN CENE 0 0NC VA1
8ANC PHU0NC PHAP V| 1|EM
CHUEN CENE 0 0NC VA1
L|EU PHAP CENE 0 0NC VA1 :
W Chuyen gene c te boc s|nh duc
dcng vot chuyen gene
W Chuyen gene c te boc s|nh ducng
dcng vot khom
CHUEN CENE
0 0NC VA1
Chuyen gene c dcng vot
thcng quo te boc E5
(Embrycn|c 5tem Ce|| te
boc mom[
CAU H0| PHAN 6
. Nhung yeu tc noc quyet d|nh t|nh doc h|eu 7 dc nhoy cuo phucng phop PCk 7
2. Lom coch noc tong t|nh doc h|eu, dc nhoy cuo phucng phop PCk 7
3. Nhung coch hon che coc nhucc d|em cuo phucng phop PCk
4. Neu mct sc ung dung cuo nested-,mu|t|p|ex, AP-PCk, kACE, |n s|tu PCk
5. Neu coch thuc h|en phon ung reo|-t|me k1-PCk
6. ucng chuon |o g| 7 Xoy dung nhu the noc 7 Dung |om g| 7
7. Ct |o g| 7 V| soc con xoc d|nh Ct 7
8. Nguyen |y cuo PCk to| tc hcp
. Nguyen |y phucng phop C|o| tr|nh tu thec 5onger, phucng phop C|o| tr|nh tu tu
dcng. Khoc b|et g|uo ho| phucng phop 7
0. Cc the goy dct b|en d|em khcng d|nh hucng nhu the noc 7
. Cc the goy dct b|en d|em d|nh hucng nhu the noc 7
2. Neu mct ung dung cu phucng phop DNA 5huff||ng
3. Coch chuyen vot ||eu d| truyen (VLD1[ c thuc vot
4. Chuyen VLD1 c dcng vot : coc coch t|ep con vo uu nhucc d|em cuo chung