Cc enzyme s dng trong

k thut to dng
1.Restriction endonuclease
2.Cc enzyme bin i DNA
Nuclease:
ExonucleaseIII,DNaseI,nucleaseS1
DNA polymerase:
DNA pol.I,Rtase,RNA pol.
Enzymes bin i u cui DNA:
Polynucleotide kinase,terminal trasferase
3.DNA ligase

I. Cc enzyme ct gii hn (RE)

1. Ngun gc
2. Phn loi

II. K thut di truyn

1. Vector to dng
2. T bo ch
3. K thut to dng

A. Chin lc to dng
B. PCR

4. Th vin gen

A. Th vin b gen
B. Th vin cDNA

Vai tr sinh hc ca RE
H thng bin i gii hn-restriction enzyme
c hot ng cng vi h thng methylase.
Methylases l enzyme thm nhm methyl vo
nucleotide chuyn bit (vo A hay C) trong
trnh t nhn bit (recognition sequence). S
methyl ha ngn chn s nhn bit ca
restriction enzyme.
Do , restriction enzyme trong mt t bo
khng phn ct DNA ca chnh n. Tuy nhin
restriction enzyme c th phn ct DNA ngoi
lai xm nhp vo trong t bo nh ca
bacteriophage.

Restriction Enzyme (RE)

c tm thy trong cc loi vi khun
khc nhau l cc endonuclease c kh
nng thy gii DNA mch i nhng v
tr xc nh
Vi khun s dng restriction enzyme
bo v chng khi cc DNA ngoi lai
Vi khun c mt c ch bo v DNA
ca chng khi hot ng ca cc
restriction enzyme ca bn thn.
Cc RE hp thnh h thng bo v
prokaryote, cha c h thng tng t
no c pht hin eukaryote.
Chng c phn lp v s dng cho cc
phng th nghim

Restriction Endonucleases
Loi I- c nhiu tiu n v,c hot tnh
endonuclease v methylase, ct mt cch
ngu nhin ti v tr cch v tr nhn bit
(recognition sequence) 1000 bp
Loi II- ct DNA ngay v tr nhn bit, khng
cn ATP, hu ht dng n phn
Loi III- c nhiu tiu n v, c hot tnh
endonuclease v methylase ct cch 25 bp
t recognition sequence

RE hot ng nh th no?

Restriction Enzyme

V tr nhn bit ca enzyme

Restriction enzyme
(endonulease) ct
DNA ti trnh t c
hiu
Nhng loi cu ni
no b RE ct?

Cu ni ng ha tr
(trong mt chui)
Cu ni hydrogen
(gia hai chui)

Cu ni
hydrogen

GGACGCTAGCTGATGAATTCGCATCGGATCCGAATCCGCTCTTTCAA
CCTGCGATCGACTACTTAAGCGTAGCCTAGGCTTAGGCGAGAAAGTT
Trnh t c hiu

GGACGCTAGCTGATGAATTCGCATCGGATCCGAATCCGCTCTTTCAA
CCTGCGATCGACTACTTAAGCGTAGCCTAGGCTTAGGCGAGAAAGTT

GGACGCTAGCTGATG
CCTGCGATCGACTACTTAA

Enzyme nhn bit 4-, 6- hoc

8- cp base, trnh t lp o
(palindromic sequence)

Cu ni ng ha tr

Hot ng ca RE

Ct

Mi enzyme ct DNA ti mt
trnh t c bit restriction
site

AATTCGCATCGGATCCGAATCCGCTCTTTCAA
GCGTAGCCTAGGCTTAGGCGAGAAAGTT

Enzyme

Ngun t vi sinh

Trnh t nhn bit

EcoRI

Escherichia coli

5' GAATTC 3
3' CTTAAG 5

TaqI

Thermus aquaticus

HindIII

Haemophilus influenzae

5' TCGA 3
3' AGCT 5
5'AAGCTT 3
3'TTCGAA 5

BamHI

Bacillus
amyloliquefaciens

5' GGATCC 3
3' CCTAGG 5

AluI

Arthrobacter luteus

5' AGCT 3
3' TCGA 5

Cc RE ph bin

EcoRI
Escherichia coli
5 nh ra

GAATTC 3
CTTAAG 5

HindIII

Haemophilus influenzae
5 nh ra

5
3

AAGCTT 3
TTCGAA 5

PstI
Providencia stuartii
3 nh ra

5
3

CTGCAG 3
CACGTC 5

Trnh t lp o

u dnh v u bng
Khi cc enzyme ct, chng c th to
ra
u dnh: mt s RE, v tr ct lch nhau
trn hai mch. Trong trng hp ny
cc u dnh c th bt cp tr li
u bng: mt s RE ct hai mch DNA
ti cng mt im, sau khi ct hai u
bng khng c kh nng t kt hp li.
ni chng li phi dng enzyme T4
ligase.

Enzyme ct

5
3

5
3

G
CTTAA

5
3

GAATTC 3
CTTAAG 5

AATTC 3
G 5

To ra u 5 nh ra (theo hng 5)
u bng: qu trnh ni sau ny
khng c hiu

5 GAA TTC 3
3 CTT AAG 5

HaeIII

HaeIII l mt restriction enzyme d trn

phn t DNA cho n khi tm thy
mt trnh t ca 4 base

5 TGACGGGTTCGAGGCCAG 3
3 ACTGCCCAAGGTCCGGTC 5
5 TGACGGGTTCGAGGCCAG 3
3 ACTGCCCAAGGTCCGGTC 5

Khi trnh t xc nh c tm ra
HaeIII s ct DNA

Nhng sn phm ny c gi
vi tn u bng = blunt ends
5 TGACGGGTTCGAGG
3 ACTGCCCAAGGTCC

CCAG 3
GGTC 5

5 TGACGGGTTCGAGGCCAG 3
3 ACTGCCCAAGGTCCGGTC 5

Tn ca restriction enzyme n t:
Loi vi khun m enzyme c tm thy
Th t m restriction enzyme c xc nh v
phn lp.

EcoRI l mt v d
Chng R ca vi khun E.coli
I l restriction enzyme u tin ca E.coli c
khm ph.

ng dng RE

Vic s dng RE c ngha quyt nh

trong s pht trin ca sinh hc phn t
eukaryote. Chng cho php ct nh b
nhim sc th khng l eukaryote

Cc RE ch yu c s dng trong vic

to dng vi mc ch to ra mt s lng
ln cc bn sao ca mt trnh t DNA xc
nh.
Chng cng c ng dng lp bn
ct gii hn (restriction map) vo vic
phn tch so snh b gen cc loi khc
nhau thng qua k thut RFLP (Restriction
Fragments Length Polymorphism Tnh
a hnh kch thc ca cc trnh t gii
hn)

Bn enzyme ct gii hn

Polymerase
DNA pol xc tc s tng hp DNA theo chiu
5 3 nh:

Cc enzyme thng dng khc

DNA pol I (Klenow pol): polylerase 5 3 , exonuclease 3 5

T4 DNA pol: ging Klenow pol nhng exonuclease 3 5
mnh hn
Taq pol: phn lp t Thermus aquaticus
Pfu pol : phn lp t Pyroccus furiosus
Reverse transcriptase:do cc Retrovirus sn sinh
DNA terminal transferase: li trch t tuyn c b

RNA pol tham gia vo vic tng hp RNA theo

chiu 5 3, thng dng nht l:

SP6 RNA polymerase: c ngun t phage xm nhim

Salmonella typhimurium
T3 v T7 RNA polymerase: c ngun gc t phage xm nhim
E.coli

Cc ligase
y l enzyme xc tc cho phn ng
ni gia hai on DNA (DNA ligase)
hay RNA (RNA ligase).
C cc ligase sau:

T4 DNA (RNA) ligase: li trch t phage T4

xm nhim E. coli
T4 polynucleotide kinase: li trch t phage
T4 xm nhim E. coli
Alkaline phosphastase: li trch t E. coli
hay t rut b

Cc nuclease
y l nhm phn ct DNA hay RNA.
Gm cc enzyme chnh sau:
DNAase I: c ngun gc t ty tng b
Nuclease S1: ly trch t Aspergillus
oryzae
Exonuclease III: tch chit t E. coli
RNAase A: hin din khp mi ni
RNAase H: dng trong tng hp cDNA

To dng phn t
(molecular cloning)
- To dng phn t dng k thut di truyn phn lp
v lm thun mt gen:
+ Tch v phn on DNA ngun
+ Gn cc on DNA b ct vo vector dng ha
+ Bin np v gi DNA ti t hp trong t bo ch: ngn
hng DNA (DNA library, DNA bank)
+ Pht hin v lm thun dng mc tiu
+ Nui cy dng mc tiu ly trch v nghin cu DNA ti
t hp

Ti t hp DNA
Ti t hp DNA s dng
restriction enzyme
Quan trng: ni hai
mnh DNA li vi nhau
chng phi c ct
bng mt loi enzyme
ct gii hn

Cu hi
Loi cu ni no do DNA Ligase to
ra?
Cu ni ng ha tr (gia nucleotide
trong mt chui DNA)

Ti sao?

Restriction enzyme v
DNA ligase c th c
s dng to DNA ti t
hp, DNA c th c ct
ni li vi nhau t cc
ngun khc nhau
To dng (Cloning) c
ngha l gn mt on
DNA (mt gen vo mt
vector to dng (cloning
vector) sau t vo mt
t bo sng (Bin np)
khuch i gen mc
tiu.

Ti sao phi to dng DNA?

S dng cc DNA c to dng l cch u tin xy
dng bn ct gii hn
phn tch gii trnh t DNA c to dng
S dng DNA to dng lm probe xc nh r mc
phin m, kch thc ca mRNA
S dng DNA to dng nh mt probe xc nh
DNA tng t trong cc loi khc nhau hoc to thnh
th vin to dng
Sn xut tht nhiu gen c to dng (V d
insulin protein)
S dng to dng to antisense mRNA cho gene
silencing
To mt reporter gene xc nh s biu hin ca
gen

DNA Cloning
Khuch i cc on DNA

Ngun DNA to dng

DNA nhim sc th (bao gm DNA
c dng v khng c dng)
mRNA c chuyn thnh cDNA
PCR- khuch i DNA mc tiu

Cng c to dng
Restriction endonuclease ct DNA
DNA Ligase ni DNA
Vector gn DNA
T bo ch khuch i DNA
Cng c chuyn DNA ti t hp vo t bo
ch
Cng c gi DNA trong t bo ch
Cng c xc nh xem t bo no c
to dng thnh cng (tm sot)

Th mang gen: vector

Mt vector cn c cc c im tiu chun sau:
C kh nng t sao chp trong t bo ch, sao chp
khng ph thuc vo t bo ch
C c tnh gip d xc nh t bo c chng
Mang nhng v tr nhn bit duy nht ca cc
enzyme ct gii hn
C kch thc cng nh cng tt, d xm nhp, sao
chp nhanhhiu qu.
C s lng bn sao trong t bo cao
Cc vector cn biu hin th phi c promoter mnh

S khc nhau ca cc vector to dng

Loi t bo m chng c th vo
Cch thc m chng xm nhp (bng bin np
hay bng cch xm nhim ca virus/phage)
ln ca on DNA ngoi lai m chng c th
mang
V tr ct gii hn chnh xc ca enzyme ct gii
hn
S n nh ca DNA khi c chn vo
Nhng kha cnh quan tm
Hiu qu to dng
S lng bn sao (trn mt t bo ch)
Kh nng tm sot (v d h thng Lac Z)

Th mang gen: vector

Vector: phn t DNA kch thc nh dng
mang gen, sao chp, thao tc trn gen
Vector to dng v vector biu hin
Vector to dng: chuyn v lu tr gen ti t
hp trong t bo ch, nhn bn, thao tc, phn
tch sau trn DNA ti t hp s d dng hn
Vector biu hin: to sn phm ca gen ti t
hp mc phin m, dch m, phn tch trnh
t iu ha s biu hin ca gen

Plasmid l cc vector to dng

V tr ct hn ch duy nht chn DNA mc tiu

(vng polylinker), c th nm ct ngang gen lacZ m
ha cho Beta-galactosidase, mt enzyme chuyn
khun lc thnh mu xanh khi hin din X-gal (khun
lc chuyn thnh mu trng khi c mt on DNA
ngoi lai chn thnh cng vo vng polylinker v lm
bt hot gen lacZ)
C promoter mnh nm hai pha ca vng
polylinker biu hin DNA c to dng.
Mt oriC
Marker chn lc (V d. Gen khng khng sinh) nu
khng c bin np vi khun s cht (chc chn rng
ch c nhng vi khun c bin np thnh cng mi
sng)

Plasmid
Plasmid l nhng on DNA ngn (2-5kb) dng
chui xon kp, dng vng, c th t nhn i c
lp vi DNA b gen
C s bn copy cao trong t bo E. coli (100)
Gen khc khng sinh (marker chn lc, VD. AmpR;
c ngha l E. coli c kh nng khng khi bin np
thnh cng nhn c plasmid)
To dng tt vi on DNA 5-10 kb (nh)
Rt d s dng

Cc loi plasmid
Plasmid th h th nht: tm thy trong t
nhin ( ColE1, pSC101)
Plasmid th h th hai: plasmid nhn to
(pBR322: kch thc 4364 bp, mang hai gen
ApR, TetR v 20 v tr nhn bit duy nht ca cc
enzyme ct gii hn)
Plasmid th h th ba: y l cc plasmid
mnh hin nay c hai c tnh c bn
Kch thc nh
C mang mt polylinker

Vector to dng l phage

+ Bacteriophage vector

c thit k i vo chu trnh tan

To dng tt vi nhng on DNA ln (10-23 kb)
Phage khng d thao tc nh plasmid

Phage c loi b 1/3 b gen ch gi li vng cha gen xm

nhp, t sao v lp ghp
DNA ngoi lai c a vo t bo ch qua phage ny, nhn bn
v lu tr cng vi phage
Vector phage cha t trnh t nhn bit ca enzyme ct gii hn

+ Thc khun th M13

+ B gen mch n c kch thc 6,4 kb

+ Thng dng gii trnh t DNA

+ Phagemid
Kt hp gia bacteriophage v plasmid
To ra + ng gi ssDNA vi s tr gip ca
phage
Hoc c th bin np vo E. coli khi n th
hin ging mt plasmid (kch thc hn ch
ging mt plasmid)
V d. pBluescript (pBS)
C promoter phage T3 v T7 trn mi hng
cho vic tng hp ssRNA in vitro

Cc vector to dng khc

+ Cosmid

Thit k nhn to bi s kt hp c u im
plasmid DNA v trnh t cos t phage
Khi vo trong t bo ch, c kh nng sao chp
nh plasmid. Plasmid ti t hp bn hn trong t
bo ch
Nhng trnh t cos cho php ng gi DNA vo
phage ti np vo t bo khc
Rt tt to dng nhng on gen rt ln
DNA (32-47 kb). Hiu sut chuyn gen cao hn
Plasmids cng ln th cng kh thao tc

BAC

Nhim sc th nhn to vi khun

Dng to dng nhng on DNA rt ln vo E. coli
(tt v n nh s dng trong to dng HGP)
C cha ORI ca nhn t f; s dng ging plasmid

Eukaryotic vector

VD. Ti plasmid ca Agrobacterium tumefaciens dng

to dng thc vt.
VD. YAC (Nhim sc th nhn to nm men) Kch
thc 10kb mang c DNA 150 1000kb. Sao chp
nh mt nhim sc th bnh thng ca nm men.
dng to bn vt l b gen v d b gen ngi
(nhng khng bn nh BAC)

10

u im ca Prokaryote
1. Pht trin nhanh
2. Thao tc d dng

T bo ch
- E. coli:
+ T bo ch ph bin nht, d nui, sinh sn nhanh
+ Vi sinh vt gy bnh c hi
+ Khng tit enzyme
- Bacillus subtilis:
+ Khng gy bnh, khng to ni c t, tit protein
+ Pasmid khng n nh trong t bo ch
- Saccharomyces cerevisiae:
+ T bo eukaryote c nghin cu di truyn k nht
+ S dng th hin gen eukaryote
- Virt ng vt (SV40, retrovirus):
+ Dng dng ha gen vo t bo hu nh, c ch
tim tan

Nhc im ca Prokaryote
1.
2.
3.
4.

Khng th tch cc intron ra

Intron cn thit cho s biu hin ca gen
Kch thc DNA a vo vi khun b hn ch
Prokaryote khng c c ch glycosyl ha
protein

Ci tin t bo ch E. coli
Thng thng E. coli dng to dng
thng c ci tin:
Loi b cc h thng sa i hn ch
bng cch gy t bin trong h thng
sa i hn ch ni sinh
H thng ti t hp DNA c sa i
ngn chn s ti t hp
Thay i hot tnh endonuclease nhm lm
tng lng plasmid tch ly trong t bo

Cc bc ca k thut to dng
1. C lp gen mc tiu, c lp plasmid DNA
2. Thao tc trn DNA
a,b. Ct - Restriction enzyme
c. Ni - DNA ligase
3. Bin np vo vi khun
4. Chn lc vi khun bin np thnh cng.
5. Xc nh dng vi khun mang gen cn to
dng

11

 Qu trnh to
dng mt gen
ngi
trong
mt vector to
dng
l
plasmid
vi
khun c th
c
chia
thnh 5 bc

Polycloning site

Gen khng khng sinh. Nu

chn vo mt gen s dng Pst
I restriction enzyme, kt qu
t bo khng tetracycline
nhng khng khng ampicillin
resistant. Ti sao?

Gen khng khng

sinh

Vector to dng in hnh

Antibiotic resistance important to help select for
organisms that are transformed with the plasmid
Copyright 2002 Pearson Education, Inc., publishing as Benjamin Cummings

Chn DNA ngoi lai

Gen ch

Bin np

S dng cng mt loi restriction endonuclease cut cloning vector v

the host chromosome. Ti sao??

Phng php khuch i v thu nhn gene t vi khun vi plasmid

Phng php khuch i v thu nhn gene t vi khun vi plasmid

Hin din cloning vector cho php chn lc trn agar c cha khng sinh
. Cc t bo khng cha cloning vector th khng c tnh khng khng sinh

Phng php khuch i v thu nhn gene t vi khun vi plasmid

12

Plasmid ti t hp

To plasmid ti t hp

Bin np plasmid
vo E. coli

Ti to dng (subcloning)

Th vin DNA

Ti to dng l kiu n gin nht ca th

nghim to dng, l qu trnh chuyn DNA
c to dng t vector ny sang
vector khc biu hin n trong mt
chng ch nht nh.

Da vo bn cht ca DNA cn to
dng ngi ta phn bit hai loi th
vin gen:
th vin b gen v th vin cDNA

Cc bc c bn ca ti to dng cng
tng t nh to dng

13

Th vin b gen (Genomic)

Th vin b gen ca mt sinh vt l tp hp
tt c cc trnh t DNA cu thnh b gen
c gn vo vector. Cc vector ti t hp
sau c a vo t bo ch. Cc t bo
ch sau c nui cy trn mi trng v
to thnh cc dng.
Th vin b gen thng c lp cho
prokaryote
B su tp cc dng vi t nht mt bn sao
cho mi on DNA c to dng trong YAC
hay BAC

Th vin cDNA
Th vin cDNA l tp hp cc bn sao cDNA t
tt c cc mRNA ca mt t bo. Nh vy khng
ging vi th vin b gen , th vin cDNA c
thit lp t mt t bo xc nh trong gen cn
nghin cu phi biu hin thnh mRNA
Thng c lp cho eukaryote
cDNA c tng hp t mRNA trng thnh
(eukaryote) (Khng c cc on intron) s dng
enzyme Reverse transcriptase, RNase H , DNA
Polymerase I, v nuclease S1.

Th vin b gen

14

Th vin cDNA

Tuyn chn dng mc tiu

C th s dng mt khng th c nh
du xc nh protein c chn
t mt vi khun lc vo mng lai; ph
v t bo
Mu d trn mng lai vi mt khng th
c hiu cho protein chn
Hoc s dng mt mu d DNA (probe) b
sung tm trc tip DNA

15

Tuyn chn dng mc tiu

c th phn lp dng t bo ch ti t
hp cha gen mc tiu trong mt th vin
DNA hay mt th vin cDNA, ngi ta phi
s dng cc phng php sng lc.
Thng thng cc phng php sng lc
c xy dng da trn vic s dng mu
d DNA.
Mu d DNA (probe) l mt on
oligonucleotide c nh du v n b
sung hoc b sung mt phn vi gen mc
tiu.

Tuyn chn dng mc tiu

- Gen c biu hin trong t bo
ch l vi khun:
+ Lai min dch (Western blotting,
Immunoblotting)
+ Hot tnh enzyme
+ B tr t bin (gen ngoi lai ng
dng vi gen ca t bo ch)
- Gen ngoi lai khng biu hin trong
t bo ch:
+ Lai insitu:
+
Lai
khun
lc
(colony
hybridization):
vector l plasmid
+ Lai plaque (plaque colonization):
vector l phage

Vector biu hin (expression vector)

- biu hin gen ngoi lai:
+ Thu protein ti t hp
+ Nghin cu cc yu t iu ha th hin ca gen

- Gen ngoi lai c kim sot bng promoter c

nhn din bi RNA polymerase ca t bo ch:
+ Promoter mnh
+ Khung dch m ng
+ Promoter thng dng: lac, trp (c ch tt m)

16

ng dng thc tin ca

k thut di truyn

ng dng ca Sinh hc phn t

Cng ngh to cc sinh vt mi c ch.

- Sn xut nhng sn phm trao i cht ca vi

sinh vt
- Sn xut vcxin phng chng virus
- Sn xut protein ca ng vt hu nh
- To ra cc thc vt, ng vt chuyn gen
- Phn lp v ng dng ngun gen vi sinh trong
x l mi trng
- To cc thuc iu ha s th hin ca gen
- Liu php gen cha tr cc bnh di truyn

Trong nng nghip

Nhng nghin cu ch yu tp trung
vo vic:
Nng cao nng sut nui trng

Cng ngh sn xut ging cy trng, vt

nui vt gii hn ca tin ha chng
chu bnh, thch nghi vi cc iu kin sinh
thi v cho nng sut cao.

To ra cc th h ng vt mi
C thm mt s tnh trng mi
Nhiu trng hn
T l nc cao hn
San pham cong nghiep

Cng ngh sn xut cc loi thc phm

mi c gi tr dinh dng cao, c gi tr
thuc dinh dng, cc cht b sung
(cc vitamin..) cc protein n bo.

17

T nhng nm 1970, cc nh khoa hc

c th chuyn mt gen l vo vi
khun v bt n biu hin gen
Tuy nhin, nhng protein phc v cho
nhu cu ca con ngi ngy cng i
hi phc tp v c th vi khun khng
biu hin c v cn n ng vt
chuyn gen

Nm 1998 c khong di 1% dc
phm l protein c tng hp ti t
hp nhng tr gi ti 12 t USD

Sn phm thnh cng u tin ca con ngi

v sn xut sn phm sinh hc thng qua
chuyn gen l Insulin v hormon sinh trng
vo vi khun (1982 v 1987)

Trong Y hoc

Ngi ta d tnh ch cn 600 con b

chuyn gen l c th cung cp nhu
cu ca c th gii v mt dc phm l
mot protein no (v d human Serum
albumin iu tr bng)

Tm hiu cc gen gy bnh nh gen gy lon

dng c tim, ung th, min dch, hng cu li
lim.

Liu php gen v liu php thay th t bo v m.

Dng phng php chuyn gen to ng vt th

nghim m b loi b hoc kho cc gen no
xc nh chc nng ca gen.
To ra cc loi protein, hormon, yu t sinh trng
dng trong tr liu (ch yu trong sa v trong
huyt thanh)
Thay i cu trc gii phu, sinh l c quan, ni
tng (a mt s gen mi, loi b mt s gen)
nhm phc v mc ch cy ghp ni tng.

18

Phng php chun on bnh mi

K thut to dng(cloning) v c bit
l k thut PCR a n k thut chun
on mi bng lai DNA v RNA.
Phng php ny hin ang c ng
dng rng ri trong chun on cc
bnh di truyn, quan h dng h, ti
phm hnh s.

Mt s thnh tu Vit Nam

1997, hon thin quy trnh cng ngh
chuyn gen hormon sinh trng ngi
vo c vng (Carassius auratus)
Nm 2003, Vin Sinh hc nhit i
chuyn gen Bt khng su vo cy
thuc l (Nicotiana tabacum) v cy
ng (Zea mays)
Nm 2005, Vin Sinh hc nhit i
chuyn gen hormon sinh trng ngi
vo c chp (Cyprinus carpio)

19