Cc Enzyme s dng trong cng ngh di truyn

Cc Enzyme s dng trong

cng ngh di truyn
Bi:
Bi Ch Bu
Sinh hc phn t pht hin v hiu r c ch tc ng ca hng lot enzyme, nh
s dng chng nh nhng cng c hu hiu trong vic ct ni v ghp cc gen.
Cc enzyme s dng trong cng ngh di truyn c th chia lm bn nhm ln sau y
- Cc enzyme gii hn
- Cc nuclease
- Cc enzyme kt ni
- Cc enzyme tng hp

Cc enzyme gii hn (RE-Rectriction Enzyme)

Hin tng gii hn v h thng hn ch - ci bin
Hin tng gii hn
Khi nghin cu v s xm nhim ca thc khun th (phage) vo t bo vi khun, ngi
ta nhn thy rng trong mt s trng hp, thc khun th khng th pht trin c
trong t bo vi khun v b my di truyn ca chng b ph hu. Ni cch khc, cc vi
khun ny khng thc khun th. Th nghim c th m t nh sau
Cho phage xm nhim hai chng vi khun A v vi khun B, cc vi khun ny c nui
cy trong hai mi trng thch hp. Sau khi thu hi v phn tch DNA ca phage, ngi
ta nhn thy:
- chng A: Xut hin nhiu DNA ca phage cn nguyn vn nhng t bo ca vi
khun b ph v (do s nhn ln ca phage trong t bo vi khun).

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Cc Enzyme s dng trong cng ngh di truyn

- chng B: T bo vi khun khng b ph v nhng DNA phage b ct thnh nhng

on c kch thc xc nh. Trong trng hp ny, DNA ca phage b mt h thng
bo v ca vi khun tiu dit khi va mi xm nhp vo vi khun.
Hin tng xy ra vi khun B gi l hin tng gii hn.
Enzyme gii hn v h thng hn ch - ci bin (Restriction modification system)
Vo nm 1962, ln u tin Werner Arber chng minh rng: C nhng enzyme c
bit, hot ng trong t bo vi khun, chng c kh nng phn bit DNA ca mnh v
DNA l ca phage. Cc enzyme ny hn ch kh nng sinh sn ca phage trong t bo
vi khun bng cch phn hy chng mt cch c hiu.
Vo nm 1970, Hamilton Smith pht hin vi khun Haemophilus influenzae Rd
enzyme gii hn v t tn cho n HindII.
Vi nhng th nghim tip theo, Arber v cng s ti trng i hc Geneva pht
hin ra nhiu chng vi khun c kh nng phn hu c DNA t loi khc xm nhp
vo chng nh kt hp ca hai qu trnh enzyme.
Hai qu trnh enzyme c thng nht trong mt h thng gi l h thng hn ch - ci
bin, nhm phn hy DNA l v bo v DNA ca mnh.
- S hn ch c thc hin nh s hot ng ca cc enzyme ct hn ch (RE Rectriction Enzyme). chnh l cc odesoxyribonuclease c bit c th nhn bit mt
trnh t nucleotide c hiu trong chui DNA si i v ct c hai si DNA .
- S ci bin l s thay i DNA ca bn thn t bo theo con ng c bit ca mi
loi, khin DNA khc vi DNA ca loi khc v khng phi l c cht ca enzyme
ct hn ch. Nh m DNA ca t bo ch c bo v. S ci bin c thc hin
nh cc enzyme metylase (mtyl ho) c trong t bo vi khun. Enzyme ny s metyl
ho cytosine v tr (C5) v adenine (trn N ca C6) thuc vng gii hn (chui ch).
Enzyme Hind III, sau khi c metyl ho (du chm) adeninee, chui ch ny
khng c nhn bit bi enzyme Hind III na, do , DNA khng b ct

Enzyme Hind III

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Cc Enzyme s dng trong cng ngh di truyn

Vy enzyme hn ch v enzyme ci bin c cng mt i tng nhn bit l chui

nucleotide c hiu trong DNA ngoi lai v DNA ca t bo ch, nhm hn ch s
sinh sn ca DNA ngoi lai v bo v DNA ca mnh. Tuy nhin, cng c mt s loi
enzyme c c hai chc nng hn ch v ci bin.

Enzyme EcoRI vi chui ch

Cch gi tn ca enzyme gii hn

Tn gi ca enzyme gii hn thng c 3 hoc 4 ch xut pht t tn ca vi sinh vt m
t , enzyme c xc nh v chit tch.
- Ch ci u (vit hoa) l ch u ca tn ging ca vi khun t enzyme c trch
li.
- Ch th 2 v th 3 (vit thng) l ch u tn loi ca vi sinh vt.
- i khi c ch th 4 ch chng sinh vt.
- Ch s la m ch th t RE c pht hin (c th c nhiu RE c pht hin t mt
chng).
V d nh EcoRI - c xc nh u tin E. Coli v EcoRV -l enzyme th 5 cng
c xc nh E. Coli.

EcoRI

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Cc Enzyme s dng trong cng ngh di truyn

Hpa I, Hpa II, Bam HI

Cc loi enzyme gii hn

Loi I
Nhng enzyme gii hn loi I c trnh t nhn bit c hiu. V tr ct ca cc enzyme
loi ny nm ngoi trnh t nhn bit mt khong cch khng nht nh, dao ng t
1.000 n 5.000 nucleotide. Do khng c tnh c hiu trong ct on nn sn phm ca
n khng ng nht v khng pht hin c bng in di trn gel.
Phn t lng ca chng vo khong 300.000D, cc bn n v khng ging nhau. Cn
Mg+2 lm cofactor v ATP cung cp nng lng cho enzyme ny chuyn ng dc
theo phn t DNA t im nhn bit n im ct. V d nh EcoK do Meselson v
Yuan tch c, ct im cch trnh t nhn bit 1.000 nucleotide hoc xa hn na.
Loi II
Bao gm nhng enzyme c trnh t nhn bit chui nucleotide c hiu. Khi nhn bit
c trnh t , chng ct ngay ti v tr nhn bit.

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Cc Enzyme s dng trong cng ngh di truyn

Phn t lng thp hn loi I v khong t 20.000 n 100.000D. Ch cn Mg+2 lm

cofactor v khng cn nng lng ATP. Do c tnh c hiu v ct on DNA nn sn
phm thy phn l tp hp nhng on DNA c hiu v cho php pht hin bng in
di trn gel agarose.
Ngy nay c trn 1.000 RE loi II c tch ra t cc t bo procaryote khc nhau v
c hn 70 loi ang c thng mi ha trn th trng.
Loi III
Bao gm nhng enzyme sau khi nhn bit mt trnh t DNA c hiu th ct v tr
cch 20 nucleotide v to ra mt s u ct khc nhau. Tuy rng v tr im ct rt
gn trnh t nhn bit ca chng nhng kh on trc c cc im ct . V vy m
RE loi III cng nh RE loi I khng c s dng rng ri trong cng ngh di truyn.
Trong 3 loi RE ni trn ch c RE loi II c ng dng nhiu hn.
Cc loi RE trong nhm II
Tnh c hiu v tr
Trnh t nhn bit ca RE
C th ni cc RE ny l nhng cng c thc th ct cc DNA. S ct ny xy ra
mt vng c bit c nhn bit bi enzyme. Mi mt enzyme nhn bit mt on
nucleotide khc nhau rt c hiu i vi n v c gi l trnh t nhn bit (chui
ch) hay on c ngc xui.
c trng quan trng nht ca cc trnh t nhn bit l chng c cu trc i xng
nghch o (palindromic), ngha l hai mch ca trnh t hon ton ging nhau khi chng
c c theo chiu 5 n 3 mi si n. Cc trnh t nhn bit ca enzyme gii hn
c cu to t 4 n 6 i baz. V d nh on DNA c ng khung sau

on DNA c ng khung

Cc kiu ct ca RE
Ct u bng: Mt s RE to vt ct trn phn t DNA ngay chnh gia palindrom, to
ra hai on DNA u bng. Sau khi ct, hai u khng c kh nng t kt hp tr li.

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Cc Enzyme s dng trong cng ngh di truyn

Hpal

Ct u dnh (u lch): Ct bn ny v bn kia ca tm i xng to ra hai u

lch nhau mt vi baz. Trong trng hp ny, cc u dnh b sung c th b bt cp
tr li. Nh vy, khi cc DNA khc ngun nhng cng c cha trnh t nhn bit c
hiu ca mt RE, sau khi b ct s c cc u dnh b sung ging nhau nn cc on
DNA khc ngun c th ni li to ra nhng phn t lai. y l c s ca phng
php to dng gen, mt phng php thc y s pht trin ca cng ngh di truyn.

EcoRi

S lng on ct
Mt RE c kh nng thao tc trn ton b chiu di ca mt phn t DNA. S lng
on ct ph thuc vo s chui ch ca mt RE c trn phn t DNA m n thao tc.
S dng RE khc nhau, s b ct khc nhau v to ra lng on ct khc nhau.

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Cc Enzyme s dng trong cng ngh di truyn

EcoRi

Nu chui ny c 5 ln trong mt phn t DNA th enzyme EcoRI s ct ti 5 im trn

phn t DNA
- Ta s c 5 on nu DNA dng vng s- Ta s c 6 on nu DNA dng thng
Kch thc on ct
V vng gii hn c phn b mt cch ngu nhin dc theo mt phn t DNA m
c to thnh bng cch t hp 4 loi baz nit (A,T,C,G) nn kch thc mi on
ct ph thuc s cp baz ca mi chui ch.
Nu vng nhn bit (chui ch) ca enzyme l 6 cp baz s sinh ra cc on c kch
thc trung bnh 4 4 = 4.096 baz 4,1kb.
Nu vng nhn bit ca enzyme l 4 cp baz s sinh ra cc mnh c kch thc l 4 4 =
256 baz 0,26kb.
Tuy nhin mt s RE nhn bit cc vng rt him gp trong DNA. V d nh NotI c
chui ch (GC\GGCCGC), khi ct cho cc mnh c kch thc khong 1.000kb. PvuI
c chui ch (CGAT\CG) cho mnh khong 300kb.

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Cc Enzyme s dng trong cng ngh di truyn

Mt s enzyme hn ch (HC), enzyme ci bin (CB) v chui ch ca n

Nhng enzyme c cng chui ch, enzyme no tm thy trc c trong ngoc ( )
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Cc Enzyme s dng trong cng ngh di truyn

iu kin hot ng ca RE
a s enzyme bn trn th trng c hot ng t 1 n 100 n v trn l. Mt n
v c tnh bng khi lng enzyme ct hon ton 1 g DNA trong 1 gi vi
th tch phn ng 50 l, nhit phn ng 37C.
Tuy nhin trong thc t, mun ct hon ton lng DNA cn phi tng nng ca
enzyme t 5 n 10 ln so vi cch tnh n v ch nh. iu kin phn ng ct ti
u l
- C Mg+2 lm xc tc
- Nhit 37C
- pH t 7,2 n 7,8
- Thi gian phn ng t 1 n 2 gi.
Cc RE b c ch trong cc iu kin: nng EDTA cao qu hoc s c mt ca phnol
(khi tch DNA cha loi ht).
ng dng ca RE trong cng ngh di truyn
Phn lp gen
Cc RE cung cp mt phng php mi c hiu lc cho vic phn lp gen. V nguyn
tc mt b gen DNA phc tp c th b ct thnh nhiu phn on, m t ngi ta
c th phn lp mt phn on mang gen c hiu bng cch in di trn gel. Nhng
bng in di DNA c hiu c th nghim cho gen nh nghin cu bng cch lai ha
vi nhng bn sao phng x ca gen tinh ch t t bo bng nhng bin php thch hp.
Gen ton b hoc mt phn ca n c th thu c nhng phn on nh hn bng cch
ct on lin tip vi nhng RE khc. T ta c th phn lp c gen mong mun.
Mun phn lp vng iu ha lac, u tin ct DNA cha gen lac bng Hind III v to
nhiu phn on trong c mt on cha lac di 660bp. on ny c tch bng
cch in di trn gel. Phn on thu c ct tip bng enzyme Hae III thnh on cha
174bp vn cn cha h thng iu ha v gen lac. Nu tip tc ct bng nhng RE thch
hp ta s phn lp c vng iu ha gen lac.
Ngoi ra, cc RE cn c s dng to cc vector chuyn gen.

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Cc Enzyme s dng trong cng ngh di truyn

Cc loi Nuclease
Phn loi
Cc nuclease c kh nng phn ct phn t DNA hoc RNA. C ba kiu phn loi cc
nuclease sau:
Da vo bn cht ca c cht m n tc dng, ngi ta chia nuclease lm hai loi
- Deoxyribonuclease (DNase), c cht ca n l DNA
- Ribonuclease (RNase), c cht ca n l RNA
Da theo kiu lin kt m enzyme thy phn, ngi ta cng chia ra lm hai loi
- Nuclease "a": Thu phn c hiu lin kt phosphodiester gia cacbon (C3) v nhm
phosphate.
- Nuclease "b": Thu phn c hiu lin kt phosphoester gia cacbon (C5) v nhm
phosphate (Hnh 4-1).
Da theo hot tnh enzyme, ngi ta cng chia ra lm hai loi exonuclease v
endonuclease
- Exonuclease: Ct cc lin kt phosphoester t hai u ca chui poly- nucleotide v
t ct dn tng nucleotide, chng i hi l phi c nhm OH (3) hoc OH (5) t
do hai u chui.

Exonuclease

- Endonuclease: Thng thu phn lin kt phosphoester v tr bt k bn trong chui

v to ra cc oligonucleotide.

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Cc Enzyme s dng trong cng ngh di truyn

Phn loi nuclease theo lin kt b ct

Mt s nuclease thng dng v ng dng ca n

DNase I
L mt endonuclease c kh nng phn ct phn t DNA si n hoc si i sau cc
baz pyrimidine to hn hp oligonucleotide c nhm phosphat u 5. Kh nng
phn ct si n hoc si i trong phn t DNA ph thuc vo s c mt ca Mn+2 v
Mg+2.
- Khi c mt ca Mn+2, enzyme ny tn cng vo si i to vt ct u bng hay u
dnh.
- Khi c mt ca Mg+2, n tn cng vo mt si trn phn t DNA mt cch c lp.
Cc DNase c s dng loi DNA ra khi dch chit RNA nhm lm tinh sch
RNA. Ngoi ra, ngi ta cn dng to vt t ngay trn phn t DNA trong k thut
nh du hay pht hin nhng gen ang hot ng trn nhim sc cht, v nng
thp chng ch ct nhng v tr siu nhy cm nh nhng gen m, vng va phin m.

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Cc Enzyme s dng trong cng ngh di truyn

Nuclease S1
L enzyme c tch t nm si Asp. oryzae, c kh nng phn hu DNA si n, DNA
si i. Khng c kh nng phn ct phn t lai DNA- RNA.
Cc enzyme nuclease S1 c s dng nhm cc mc ch sau
- Phn tch cu trc ca cc phn t lai DNA- RNA,
- Loi b cc mch n ca u so le to u bng,
- Loi b nhng cu trc kp tc trong khi to cDNA.
Desoxyribonuclease III (Exonuclease III )
Enzyme ny c tch t vi khun E. Coli. N xc tc phn ng phn ct cc trnh t
nucleotide t u 3OH t do ca mt theo hng 3 5. Kt qu l to vng mch
n di trn DNA si i, ngoi ra cn c hot tnh 3 phosphate.

Desoxyribonuclease

Enzyme ny c ng dng to cc cu trc mch n mt s vng trn phn t

DNA sn xut mu d c trng cho tng mch hoc phi hp vi nuclease S1 to
t bin mt on ti nhng vng c bit.
Enzyme ribonuclease ( RNase A v RNase H)
RNase hin in mi ni, enzyme thng mi c trch ly t ty b.
c trng ct lin kt phosphodiester ngay sau mt baz pyrimidine nh uracil v
cytosine ca RNA mch n. RNase A bn nhit khng mt hot tnh 90C trong mt
gi.
c ng dng loi b RNA cn st li trong dch chit DNA hoc loi b cc vng
khng bt cp ca RNA trn phn t lai DNA- RNA.
RNase H l enzyme xc tc loi b RNA. Sau khi phin m ngc tip tc tng hp
mch th hai ca cDNA hnh thnh mch DNA si i.

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Cc Enzyme s dng trong cng ngh di truyn

Enzyme phosphodiesterase
L mt loi nuclease c trong nc rn, c cht tc dng l DNA v RNA, tn cng t
u 3 ca chui polynucleotide.

Emzyme kt ni
Ligase
kt ni hoc hn hai mnh DNA hoc RNA ngi ta phi s dng enzyme ligase
trong s c mt ca ATP. Khi to ra mt lin kt ester gia mt mnh cha 5
phosphat v mt mnh cha 3OH. Chng thng c s dng kt hp vi enzyme
khc nh kinase v alkaline phosphatase. Thng thng, chp hai mnh u dnh d hn
hai mnh c u phng.
Ecoli DNA ligase
Enzyme ny c trch ly t E. Coli, ch c th xc tc ni hai mnh DNA c u so le.
ng dng to DNA ti t hp trong k thut tch dng.
T DNA ligase
4

Enzyme ny c trch t phage T . C kh nng xc tc ni hai trnh t DNA u so

4

le v c bit l hai trnh t u bng. Kh nng gn mnh hn 10 ln so vi ligase tch

t E. Coli. c a chung trong k thut tch dng.
T RNA ligase
4

Enzyme ny c trch t phage T , xm nhim E. Coli. C kh nng xc tc ni hai

4

trnh t RNA bng lin kt phosphodiester. C cht ca n thng l nhng phn t nh

nn c dng nh du phng x u 3 ca cc phn t RNA dng lm mu d phn
t.
Cc enzyme dephosphoryl ho ( kh nhm PO4

Alkaline phosphatase
c trch ly t E. Coli hay t rut b. c tnh ca enzyme ny l hot ng pH kim
v c kh nng xc tc kh nhm phosphate 5 ca mt chui DNA hoc RNA v cc
nucleotide t do.

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Cc Enzyme s dng trong cng ngh di truyn

Phn ng ni DNA ngoi lai vi vector kh nhm PO4-3

Alkaline phosphatase c ng dng

- Loi b nhm phosphate 5 trn trnh t DNA hoc RNA trc khi nh du phng x
5 ca chng bng P32 lm mu d phn t lai.
- Loi b nhm phosphate 5 trn mt vector va mi b ct bi enzyme RE. Nhm
trnh vector ny ng kn tr li, khin cho vic ci mnh DNA ngoi lai vo b cn tr.
Trong trng hp ny, si kp c ci vo c mang hai nhm phosphat 5 s to ra
hai trong bn lin kt este hnh thnh mt DNA ti t hp kp vng kn c cha hai
khe h.
S chp ni vector b kh PO4

5 vi DNA ngoi lai to ra DNA ti t hp

si kp c hai ch trng (OH) do OH 3 ca on ngoi lai khng to lin kt ester vi

OH 5 ca vector khhm PO4
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Cc Enzyme s dng trong cng ngh di truyn

Cc enzyme phosphoryl ho
- Enzyme kinase (T4 polynucleotide kinase): c chit t trc khun T4 xm nhim
E. Coli. C kh nng chuyn nhm phosphate t phn t ATP ln u 5 ca DNA hay
RNA b kh phosphate bi enzyme alkaline phosphatase.
Enzyme kinase c ng dng nh du phng x u 5 ca DNA lm mu d
phn t trong k thut lai hoc chuyn nhm PO4 3 ti cc trnh DNA khng c
nhm PO4 3 trong phng php to dng.

Cc Enzyme tng hp
Enzyme sao chp mt axit nucleic: Qu trnh sao chp chui axit nucleic tc l tng hp
mt chui DNA hoc RNA c tin hnh theo nguyn tc b sung v i song song.
Vic thm mt nucleotide mi c tin hnh theo hng 5 3.
Enzyme sao chp DNA DNA
L cc enzyme DNA-polymerase I, enzyme T4 DNA-polymerase, Taq polymerase tng
hp chui DNA t mt khun DNA hay cn gi "enzyme ph thuc DNA".
Enzyme DNA - polymerase I
Enzyme DNA-polymerase c ngui gc c tch t vi khun E. Coli, chng c ba hot
tnh
- Hot tnh tng hp DNA theo hng 5 3 v sa cha trong sao chp,.
- Hot tnh exonuclease theo hng 3 5,
- Hot tnh exonuclease theo hng 5 3.
Ngy nay enzyme DNA-polymerase c dng ch yu xc nh trnh t DNA bng
phng php didesoxynucleotide ca Sanger, ngoi ra, cn dng tng hp mu d c
phng x cao, hoc xy dng cc vector t DNA mch n.
Trong thc t ngi ta hay s dng on Klenow l sn phm thy phn ca enzyme
DNA-polymerase I, c hot tnh exonuclease theo hng 3 5 v hot tnh tng hp.
Enzyme T4 DNA - polymerase
C ngun gc t phage T4 xm nhim E.Coli c hot tnh exonuclease theo hng 3
5 . Enzyme ny mnh nn c s dng nhiu tng hp mu d c phng x
cao.
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Cc Enzyme s dng trong cng ngh di truyn

Enzyme Taq - polymerase

Trch li t vi khun Thermocellus aquaticus, l enzyme chu nhit cao, c tc dng
khuych i tng hp DNA. Enzyme ny ch yu s dng nhn dng gen trong phn
ng PCR.
Enzyme phin m ngc (Reverse transferase)
L enzyme c kh nng sao chp b gen RNA ca retrovirus khi k sinh trong t bo
ch to ra cDNA, theo chiu 5 3 cn c mt ca mi. y l giai on cn thit
to ra ngn hng cDNA.
Hin nay trn th trng enzyme phin m ngc c ngun gc t AMV (Avian
Myeloblastosis Virus). Enzyme phin m ngc l mt DNA- polymerase 5 3, c
cc c tnh sau:
- L enzyme ph thuc RNA,
- Tng hp DNA theo hng 5 3,
- C hot tnh RNase.
Chng c ng dng thit lp ngn hng cDNA hoc tin hnh phn ng PCR trn
mRNA, ngoi ra, chng cn c s dng xc nh trnh t DNA bng phng php
s dng cc didesoxynucleotide ca Sanger.
Cc enzyme tng hp RNA (RNA-polymerase)
C ba loi RNA polymerase c s dng nhiu nht hin nay l SP6 RNA-polymerse
c ngun gc t phage xm nhim Salmonella typhimurium. T3, T7 RNA-polymerase
c trch ly t phage T3 v T7 xm nhim E. Coli.
Cc enzyme ny hot ng trn si khun DNA xc tc s tng hp RNA theo hng
5 3. Qu trnh tng hp khng cn mi nhng khun DNA phi mang promoter c
trng ca phage. Cc enzyme ny c ng dng :
- tng hp cc mu d RNA nh du phng x trong phng th nghim.
- Nghin cu bn phin m RNA ca mt DNA c dng ha nh phng php
PCR.
- Xc nh trnh t DNA c gn trong mt vector c mang cc promoter c trng
ca phare SP6, T3 , T7.

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Cc Enzyme s dng trong cng ngh di truyn

Ngoi ra ngi ta cn dng enzyme ny tng hp lng ln RNA t DNA c ni

ngay sau promoter thch hp.
Enzyme terminal - transferase
Enzyme ny c trch ly t tuyn c b. C cc c tnh:
- Terminal-transferase xc tc gn cng mt loi nucleotide vo u 3OH t do ca
phn t DNA to ui polynucleotide.
Chng c ng dng
- Thm ui polynucleotide to u sole cho phn t DNA dng trong k thut to
dng.
- nh du u 3 (OH) ca phn t DNA xc nh trnh t axit nucleic theo Manxam
v Gilbert.

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