K thut sc k Chromatography Technology Cc k thut phn tch trong
Cng ngh Sinh hc Thc phm
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Nguyn tc ca phng php sc k
Nguyn tc ca phng php sc k
S phn tch cc cht tan da vo i lc tng i ca: cht tan i vi pha di ng
v cht tan i vi th nn. Mt cht tan c i lc i vi pha di ng ln hn so vi
i lc vi th nn s c xu hng di chuyn cng pha so vi pha di ng. Trong mt
dung dch c n cht tan, cht no c i lc vi pha di ng ln nht s c thi
gian lu nh nht, s ra khi ct sc kh sm nht.31/10/2011 8:58 SA 3 Nguyn
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Cc phng php sc kSc k hp thc (Adsorption chromatography) Sc k
trao i ion (Ion-exchange chromatography) Sc k lc gel (Gel
permeation chromatography) Sc k i lc (Affinity chromatography) Sc k
o pha (Reverse phase chromatography) Sc k lng cao p (High
performance liquid chromatography)
Ky thuat sac ky Mu (sample): cn c lc trc khi chy sc k. Buffer:
lc nhm loi b tp nhim trc khi s dng. Pha ng (mobile phase): Dung mi
bm t reservoir vo ct (column) sc k. Nhiu hn mt reserovoir, pha ng c
bm vo Mixer (my trn) trc khi vo ct. Pha tnh (stationary phase): c
gi li trong ct bi mt ming bng thy tinh y ct. Cc phn t sinh hc c tch
ra ti ct sc k. Trong HPLC, Guard column c s dng nhm bo v ct sc k.
Detector: UV bc sng 280 nm detect hu ht protein UV 205 220 detect c
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Sac ky long (liquid chromatography)
Thnh phn c bn Partition coefficient:
Cs: nng mu trong pha tnh (Stationary phase) Cm: nng mu trong pha
ng (Mobile phase)
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Sac ky long
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Thng s trong sc k lng Retention time (tR): thi gian phn t sinh
hc ra khi ct sc k tnh t lc np mu. retention volume(VR):
Thng s trong sc k lng Resolution (R): Thng s dng nh gi mc tch cc
phn t sinh hc trong mu.
Rf
W: rng ca y beat.
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Pha tnh (stationary phase)
Tch ra khi ct (elution)
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Mobile phase: ng chm, c pH, lc lin kt ion, tnh phn cc thay i
theo dy. Gim h s K. Mi phn t sinh hc trong mu c kh nng i lc ring i
vi pha tnh. 1431/10/2011 8:58 SA Nguyn Hu Tr
Open column chromatography (low performance liquid
chromatography) S dng p sut kh quyn, phn tch thp ng knh ca cc ht
trong pha tnh tng i ln. Ct s dng cc vt liu r tin. Vd: plastic. -
phn tch thp.
Sc k lng hiu nng cao (High performance liquid chromatography
HPLC) Phase ong c bm vao he thong vi toc o dong chay cao (high flow
rate) bi ap suat cao (55 Mpa). ng knh cac hat trong pha tnh nho,
cha lo nho li ti giup cac phan t sinh hoc i vao, va ac biet chu c
ap suat cao (cac hat phai ran hn cac hat c dung trong open column
chromatography) Viec tach ra tot khi toc o pha ong on nh. Trong
HPLC, pha ong thng la hon hp cua hai thanh phan hoac nhieu hn ->
mixer. ng ong va cot sac ky c lam bang thep khong r. Dung moi phai
c kh kh va loc trc khi s dung.16 Nguyn Hu Tr
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S h thng HPLC
Sc k lc gel (gel filtration) Size exclusion chromatography hay
gel filtration Phn t sinh hc c gi trong pha dung mi sut qu trnh sc
k. Tch lc cc phn t sinh hc da trn kch thc v hnh dng.
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Sc k lc gel (gel filtration)
Protein nh vo cc ht bead v c gi li. Ct thy tinh Cc ht gel
polysaccharide. Cc protein ln b lai ra khi ht v ra khi ct sc k trc
protein nh hn.19 20
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Sc k lc gel (gel filtration) Pha tnh gm cc ht gel cha nhiu khong
khng nh (xp). Protein cng nh, cng d vo ht gel v c gi lu hn. Protein
tch ra khi ct theo th t khi lng phn t gim dn.
Sc k lc gel (gel filtration) ng dng: Xc nh khi lng phn t Loi b
cc phn t c kch thc nh khng cn thit. Vd: Loi b mui khi dung dch
protein trc khi thc hin sc k trao i ion. Tinh sch protein
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Sc k hp ph Trong sc k hp ph, cht tan v pha ng cng tranh nhau v
tr gn trn pha tnh. Pha tnh c cu trc m gip protein d dng thm nhp vo
cc ht tin v v tr gn kt (binding site) Ty vo kiu lin kt gia protein
v pha tnh: Sc k trao i ion Sc k i lc Sc k k nc Sc k ngc pha23 Nguyn
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Sac ky trao oi ion
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Sac ky trao oi ion Protein thay th counterion Na+, Cl gn vo pha
tnh bng lc ht tnh in. Khi Na+, Cl- c gi l Ion exchange group hay
ion Exchanger. S gn kt hon ton-> trung ha in
in tch trn protein thay i theo pH mi trng in tch protein do in
tch cc mch nhnh (R side chain) cu t mi trng pH nht nh quyt nh. Ti
pH acid, hu ht Protein tch in dng v ngc li.
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Cc nhm tch in
Ion exchanger C 2 loi ion exchanger: Cation exchanger: c kh nng
gn kt ion dng Anion exchanger: c kh nng gn kt ion m Mch nhnh tch in
gn vo: cellulose, Agarose, dextran, hay silica (HPLC)
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Mot so ion exchanger thong dung
Tch ra Gradient nng NaCl v KCl c dng tch ra protein ra khi ct sc
k. (Cng c th dng gradient pH ca buffer)
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Sc k k nc (hydrophobic chro.) Non polar side chain: Ala, Val,
leu, Ile, Trp, Met, Phe, Pro. Thng tp trung ti li protein. C th tm
thy trn b mt protein cc protein khc nhau c tnh k nc b mt khc nhau.
c s ca sc k k nc.
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Cc loi ligand
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Tch ra Protein c np vo ct sc k cha nhm octyl v phenyl vi s hin
din 2M (NH4)2SO4. Tch ra: dng gradient nng gim dn (2M 0)
(NH4)2SO4
Normal phase chromatography Pha tnh: phn cc. Pha ng: gradient
nng dung moi phn cc tng dn. Protein phn cc hn s ra khi ct sau v ngc
li.
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Sc k ngc pha
Sc k ngc phase (reversse phase chro.) Pha tnh: cc nhnh
hydrocarbon (C-4, C8, C-18) c gn ln pha tnh. Mu c cho vo dung mi
phn cc nh: nc, methanol, acetronitrilehoc hn hp. Proteins s gn vo
pha tnh theo tng tc k nc theo cc mc khc nhau. Tch ra: s dung
gradient nng tng dn acetonitrile v gradient nng nc gim dn -> s
tng dn ca tnh k nc trong pha ng
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Sc k i lc (affinity chro.) Enzyme nhn bit v gn vi c cht l mt dng
i lc. -> l thuyt xy dng sc k i lc. Pha tnh: Affinity ligand c c
trn pha tnh.
Sc k i lc (affinity chro.)
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Mt s v d sc k i lc Antigen ligand -----antibody Protein A c c nh
trn pha tnh. IgG (immunoglobulin)s gn vi protein A ti vng Fc: nng
mui cao, Ph cao. Tch ra: pH 2-4; IgG 1; IgG 2a; IgG 2b c phn tch ra
khi ct da vo s khc nhau ca vng Fc.
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Qui trnh ELISA
31/10/2011 8:58 SA Modified from Specter, S. C., R. L. Hodinka
and S. A. Young. Clinical Virology Manual, Third Edition . ASM
Press, 2000.
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31/10/2011 HIV ELISA test. Figure 5.20: 8:58 SA
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Hank Morgan/Science Photo Library/Photo Researchers, Inc.
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K thut ELISA gin tipMu xt nghim Cng hpE E
K thut ELISA SANDWICHMu XN
C cht
Ngng phn ng. c kt qu
R A
R A
KN gn trn ging
R A 3 X
+
E Cng hp
R A 5 X
+c cht
Ngng phn ng. c kt qu 450nm
37 C
37 C
30 pht, T thng
30 pht/ 37 C
30 pht/ 37 C
30pht/ To phng
Phan ng mau ty le thuan vi nong o khang the31/10/2011 8:58 SA 49
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K thut ELISA cnh tranhMu xt nghim
K thut ELISA pht hin ng thi Khng nguyn v Khng th
Y Y?Mu XN
Y
E
R A
Ngng phn ng. c kt qu
E R A 5 X
E Cng hp 37 C
C cht30 pht, T thng
KN & KT
R A 3 X
+Cng hp
+c cht30pht/ To phng
E
Bc 1: 60 pht/ 37 C
Bc 2 : 30 pht/ To phng
Phan ng mau ty le nghch vi nong o khang the31/10/2011 8:58 SA 51
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Tch chit nucleic acid - nh tnh v nh lng c bn1. Cc phng php tch
chit nucleic acid Phng php tch chit DNA Phng php tch chit RNA ton
phn v mRNA Ly tm Phng php sc k 2. Cc phng php nh tnh v nh lng th
nucleic acid Phng php o bng quang ph k Phng php in di31/10/2011
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Tch chit DNA
thu nhn DNA tinh sch cn loi b nhng thnh phn tp nhim, m quan trng
nht l protein. S tch chit DNA da trn nguyn tc ha tan khc nhau ca cc
phn t khc nhau (nucleic acid/protein) trong hai pha khng ha tan
(phenol, chloroform/nc). Mc ch l thu c cc phn t nucleic acid trng
thi nguyn vn ti a, khng b phn hy bi cc tc nhn c hc hay ha hc. Cc
nucleic acid cn c tch chit trong iu kin nhit thp c ch hot ng ca cc
enzyme ni bo (DNase v RNase). Sau cng on tch chit, nucleic acid
tinh sch nm trong mt th tch dung dch ln. S ta kt hp vi ly tm cho
php thu nhn nucleic acid di dng cn ta d bo qun v khi cn c th ha li
trong nc theo nng mong mun.31/10/2011 8:58 SA 54 Nguyn Hu Tr
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Tch chit DNA
Bc 1: ph mng t bo, mng nhn Bc 2: loi protein Bc 3: thu hi
nucleic acid
Ph mng t bo, mng nhn: nghin t bo, m trong mt hn hp cht ty (SDS)
v proteinase ph v mng t bo, mng nhn, gii phng DNA ra mi trng ng thi
phn hy cc protein lin kt vi DNA. Cht ty l phn t lng cc, s kt hp vi
protein mng v cc phn t phospholipid lm ph v cu trc mng. Cht ty ion
ha c tc dng ph mng mnh, cht ty khng ion ha c tc dng ph mng nh hn.
Loi protein: lc mu trong dung dch phenol:chloroform bin tnh protein
ng thi khng ha tan nucleic acid. Protein b bin tnh khng ha tan
trong pha nc c cha nucleic acid v sau khi li tm s ta thnh mt lp nm
gia pha nc v pha phenol:chloroform. Thu hi nucleic acid trong pha
nc. Ta nucleic acid: nhm thu nhn nucleic acid di dng c c bo v chng
khi s phn hy ca cc enzyme v khi cn c th ha li trong nc theo nng
mong mun. C th ta trong ethanol hoc isopropanol. Sau li tm thu nhn
li nucleic acid. Cn ta c ra trong ethanol 70% loi b cc mui hoc cc
du vt ca isopropanol.31/10/2011 8:58 SA 56 Nguyn Hu Tr
Tch chit DNA
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Tch chit DNALy tm phn on (a) v phn on vng (b) phn tch cc trnh t
trong hn hp da trn khi lng ca chng. Thi gian v lc ly tm c xc nh cho
tng nhm phn t. Dung dch ly tm c t trng thp hn cc phn t cn phn
tch.
Tch chit DNALy tm ng t trng (isopycnic) phn tch cc phn t trong
hn hp da trn t trng ca chng. Thi gian v lc ly tm l chung cho cc nhm
phn t. Dung dch ly tm c t trng vi gi tr i t thp n cao hn t trng ca
cc phn t cn phn tch. Ly tm trn gradient lin tc CsCl: trong qu trnh
ly tm, dung dch CsCl s t ng hnh thnh mt gradient ng t trng vi t
trng tng dn t ming ng xung y ng. Di tc ng ca lc ly tm, cc nucleic
acid di chuyn trong ng v n v tr c t trng bng vi t trng ca chnh n s
ngng li v hnh thnh mt lp c nh trong ng. Lp ny c thu nhn li sau ly
tm.
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Phng php sc k Sc k i lc: trn poly U-Sepharose hay
oligodTcellulose, dng tinh sch mRNA. Sc k lc gel dng trong phn tch
cc nucleic acid v nucleotic t do sau qu trnh to mu d (probe) nh du.
Sc k trao i ion trn vi ct thu hi nhng lng rt nh DNA. Sc k lng hiu
sut cao: c phn gii rt cao, c dng trong tinh sch cc oligonucleotide
tng hp, plasmid, phn tch cc on DNA.
2. Cc phng php nh tnh v nh lng th nucleic acid in di:- Gel
agarose - Gel polyacrylamide
Quang ph k: o mt quang (OD Optical density)
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Phng php in di Mc tiu: nh tnh: s hin din, cu hnh phn t, kch thc
nh lng: hm lng tng i so vi thang hm lng Chun b: thu hi on DNA (dng
to dng, ) Nguyn tc: da vo c tnh cu trc ca cc nucleic acid: tch in m
ng u trn khp b mt ca mt in trng nn s di chuyn v cc dng ca in trng.
Tnh linh ng ca phn t khi di chuyn trong in trng ph thuc vo khi lng
phn t v nng cc cht cu thnh gel. Kiu in di: Agarose, polyacrylamide,
in di trong trng xung(PFGE - Pulse Field Gel
Electrophoresis)31/10/2011 8:58 SA 61 Nguyn Hu Tr
in di - Electrophoresis Chun b mu, gel (agarose,
polyacrylamide), bn in di, buffer, dung dch in di Np mu Chy in di
Nhum gel.
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in di trn gel Chun b mu, gel (agarose, polyacrylamide), bn in
di, buffer, dung dch in di Np mu Chy in di Nhum gel.
Cht nhm gel - Stain
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in di trn gel agarose Gel agarose l loi gel thng dng nht, thng
dng phn tch nhng on c kch thc 0,5-20 kb. in di theo phng nm ngang
phn gii thay i khi nng agarose hay loi agarose thay i. Pht hin bng
phng x t ghi, nhum ethidium bromide. Cht ny c kh nng gn xen vo gia
cc base ca nucleic acid v s pht hunh quang di tia t ngoi. ng dng:
pht hin 1 trnh t DNA, phn tch hn hp cc trnh t DNA (Southern blot),
chun b nguyn liu31/10/2011 8:58 SA 65 Nguyn Hu Tr
in di bin tnh (Denaturing electrophoresis) SDS polyacrylamide
gel electrophoresis (SDS PAGE): Gel: polyacrylamide SDS: Mch 12
cacbon k nc v mt u hiu nc. -> che ph phn k nc trn protein ->
protein tch in m (-).66
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in di trn gel polyacrylamide c dng tch cc on c kch thc nh, di
1000 cp base. in di theo phng thng ng phn gii cao, phn bit c nhng
trnh t ch cch nhau 1 nucleotide. Phng php pht hin: DNA phng x t
ghi, xanh methylene, ethidium bromide, protein xanh Coomassie, nhum
nitrate bc. ng dng: tinh sch cc oligonucleotic tng hp, xc nh trnh t
DNA, tch cc trnh t DNA c kch thc gn bng nhau, SDS-PAGE (phn tch
protein)
Phng php nh lng bng quang ph k Cho php nh lng tng i nng nucleic
acid c trong mu. Nguyn tc: da vo s hp thu mnh nh sng t ngoi bc sng
260 nm ca cc base. Gi tr mt quang bc sng 260 nm ca cc mu o cho php
xc nh nng nucleic acid trong mu da vo mi tng quan: Mt n v OD260nm
tng ng vi nng : 50g/ml cho mt dung dch DNA si i 40 g/ml cho mt dung
dch DNA hay RNA si n nh tnh: sch da trn t s OD260/OD280, tnh cht ca
nucleic acid (mch i/n) (280 nm l bc sng cc protein c mc hp th cao
nht.
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C s ca s lai phn t
Cc phng php lai phn t
Khi mt phn t DNA mch i c un ln mt nhit vt qu nhit nng chy (Tm)
th hai mch s tch ri nhau do s ph v cc lin kt H gia hai mch. Sau khi
hai mch tch ri, nu nhit phn ng c lm gim t t cng vi iu kin th nghim
thch hp, chng s bt cp tr li. Hin tng ny c gi l s lai phn t. c im ca
s lai phn t: c hiu tuyt i: s ti bt cp ch xy ra gia hai trnh t hon
ton b sung vi nhau. Cc trnh t b sung c th l DNA hay RNA, dn n s hnh
thnh cc phn t DNA-DNA, RNA-RNA hay cc phn t lai DNA-RNA31/10/2011
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Cc kiu lai phn t Lai trn pha lng Lai trn pha rn Lai ti ch
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Lai trong pha lng: Cc trnh t cn lai nm trong pha lng, l mt dung
dch m. S lai phn t xy ra khi cc trnh t ny gp nhau do chuyn ng nhit
v khi nhit mi trng thp hn Tm. Phng php ny c s dng pht hin cc trnh t
tng ng (gia cc loi hay trong mt c th).31/10/2011 8:58 SA 72 Nguyn
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Lai trong pha rn: Mt trong hai trnh t cn lai c c nh trn gi th rn
(mng lai). Pht hin phn t lai thng qua mu d (probe) c nh du ng v
phng x. Ba k thut lai trn pha rn thng dng l Southern blot, Northern
blot, Dot blot.31/10/2011 8:58 SA 73 Nguyn Hu Tr
Lai ti ch: Trnh t nucleic acid cn tm khng c tch chit ra khi m
hay t bo. Qu trnh lai vi mu d c nh du v pht hin cc phn t lai c thc
hin ngay trn nhim sc th, t bo hay lt ct m.31/10/2011 8:58 SA 74
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Southern blot 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. u thp nin 1970,
s ra i ca phng php in di trn gel 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 1975.31/10/2011 8:58 SA 75 Nguyn Hu Tr
Southern blotSouthern 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 ngay trn gel, DNA si kp s c tch thnh DNA si n. Ch DNA si n mi c
th chuyn ln mng lai. Chuyn DNA bin tnh ln mng lai (mng
nitrocellulose hay mng nylon). 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.
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.31/10/2011 8:58 SA 76 Nguyn Hu Tr
Northern blot Sau 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. Northern blot bao gm cc bc c bn sau: RNA (RNA tng s
hoc ch mRNA) c phn tch bng in di trn gel agarose. RNA sau khi phn
tch c chuyn ln mng lai (cc phn t RNA gi nguyn v tr nh trn gel). 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. 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 mu. 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.31/10/2011 8:58 SA 78 Nguyn Hu Tr
S m t phng php Southern blot31/10/2011 8:58 SA 77 Nguyn Hu
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Phng php PCR 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; 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. Nguyn
tc ca phng php PCR: da vo c tnh hot ng ca DNA polymerase: cn s hin
din ca nhng mi chuyn bit hot ng tng hp mt mch DNA mi t mch khun. Nh
vy, nu ta cung cp hai mi chuyn bit bt cp b sung vi hai u ca mt trnh
t DNA, ta s ch tng hp on DNA nm gia hai mi. 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.31/10/2011 8:58 SA S
m t phng php Northern blot
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Giai on bin tnh (denaturation) Trong giai on ny phn t DNA mu b
bin tnh nhit cao (thng l t 94-950C, ln hn nhit nng chy ca phn t)
trong vng 30 giy n 1 pht, tt c cc lin kt hydro gia hai mch ca phn t
b b gy v to thnh cc DNA si n.
Ba giai on trong mt chu k ca phn ng PCR
Giai on lai (hybridization) Nhit c h thp ( thng t 40-700C, thp
hn nhit nng chy ca mi c s dng khong t 3-50C) cho php cc mi bm vo cc
phn t DNA si n, nh du phn DNA cn c khuych i. Giai on ny ko di t 30
giy n mt pht. Giai on ko di (elongation) Nhit c tng ln n 720C gip
cho DNA polymerase xc tc tng hp DNA tt nht. Cng vic ca DNA
polymerase l di chuyn dc theo DNA si n v s dng n lm khun tng hp si
DNA mi b sung vi DNA mu bng cch ko di cc phn c nh du bi cc mi. Thi
gian ca giai on ny ph thuc vo kch thc ca DNA mu, thng ko 81 Nguyn
Hu Tr 31/10/2011 8:58 SA di t 30 giy n nhiu pht.
31/10/2011 8:58 SA
th biu din mi quan h gia thi gian v nhit 82 trong mt chu k ca
phn ng PCR Nguyn Hu Tr
Cc thnh phn ca phn ng PCR1. Primer (mi): Mi l nhng on DNA si n
ngn v cn thit cho vic xc tin phn ng dy chuyn tng hp DNA. Chng bt cp
b sung vi mt u ca DNA mu v to ra v tr bt u ti bn. Cc mi ny c chiu
ngc nhau, bao gm mt mi xui (forward primer) v mt mi ngc (reverse
primer). Mi l mt ch tiu quan trng ca phn ng PCR quyt nh tnh c trng
v hiu qu ca phn ng. Do vic thit k mi cn c tun th mt s nguyn tc nht
nh: di khong 18-24 base; Tm ca 2 mi gn nhau; thnh phn nucleotic ca
cc mi cn bng trnh cc cp GC lp i lp li nhiu ln; mi phi c trng cho
trnh t DNA cn khuych i; khng hnh thnh primer dimer, khng phi l trnh
t lp li. 2. DNA bn mu (DNA template): hm lng nhng khng qu cao, tinh
sch khng cha cc cht c ch phn ng (SDS, cht c ch t mu (hemoglobin, sc
t, heparin, ). PCR vn cho kt qu tt vi DNA thu nhn trc tip t t bo
hay nhng mu DNA khng c bo qun tt. 3. Nng MgCl2: cn cho hot ng ca
Taq polymerase, hm lng qu cao s to nhiu sn phm k sinh, qu thp th lm
gim hiu qu nhn bn ca enzyme. Nng ti u phi c xc nh cho tng phn ng
qua nhiu th nghim.31/10/2011 8:58 SA 84 Nguyn Hu Tr
Sau mi chu k, s bn sao ca DNA mu li c tng gp i. y l s nhn bn
theo cp s nhn. Nh vy c 1 phn t DNA mu, sau phn ng PCR vi n chu k s
to thnh 2n bn sao phn t DNA.31/10/2011 8:58 SA 83 Nguyn Hu Tr
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Cc thnh phn ca phn ng PCR4. dNTP: thnh phn 4 loi dNTP phi cn
bng, s mt cn bng lm tng cc li sao chp ca polymerase; hm lng thng
khng i nhng c th thay i ty iu kin thc t. 5. Enzyme polymerase chu
nhit Tag-polymerase l DNA polymerase chu nhit s dng cho phn ng PCR
ln u tin c bn trn th trng. Tag-polymerase c nh Vi sinh vt hc Thomas
Brock pht hin Thermus aquaticus mt loi vi khun pht trin mnh nhit
cao. Enzyme chu nhit ny gip gii quyt vn ca enzyme bin tnh sau mi
chu k. T n nay, mt s vi sinh vt chu nhit khc c khm ph v ngi ta tch
chit thm c cc DNA polymerase chu nhit s dng cho phn ng PCR nh
Vent/DeepVent DNA polymerase (Thermococcus litoralis), Pfu DNA
polymerase (Pyrococcus furiosus), Tth DNA polymerase (Thermus
thermophilus), UlTma DNA polymerase (Thermotoga maritima)... 6. S
lng chu k phn ng ty thuc vo s lng bn mu ban u, thng thng s lng chu
k nh hn 40 cho mt phn ng PCR. Thi gian ca tng chu k ph thuc thit b
v kch thc sn phm. 7. Thit b v dng c cho phn ng PCR: cn p ng c nhu
cu thay i nhit tht nhanh v chnh xc. Nhng ci tin my lun nhit quan tm
n microtiler plate 96 ging, cc block nhit ring bit trong 1 my,
block dng cho lam knh tin hnh in situ PCR.31/10/2011 8:58 SA 85
Nguyn Hu Tr
Cc ng dng ca phng php PCRHin nay thnh tu ca PCR m ra nhiu trin
vng cho sinh hc phn t, vi nhiu ng dng trong sinh hc, trong y khoa,
trong nng nghip, Mt s ng dng c tnh tng qut: Sn xut mu d: PCR gip sn
xut nhanh mt lng ln mu d nh du khi thc hin cc phn ng vi cp mi chuyn
bit v cc nucleotide nh du. Khuych i s lng cc RNA: s dng k thut phi
hp RT-PCR (kt hp enzyme phin m ngc v Taq polymerase; hoc s dng Tth
polymerase). RT-PCR cho php nghin cu cc mRNA thng tin tn ti vi hm
lng rt thp, khng th pht hin bng phng php c in nh Northern blot, K
thut in situ RT-PCR cho php khuych i cc RNA ngay trn m v t bo. nh
lng bng phng php PCR: dng nghin cu cc trnh t RNA hay DNA c s lng bn
sao rt thp. Trnh t ch c khuych i ng thi vi mt trnh t chng c nng
bit, sau phn ng so snh hm lng sn phm, v suy ra s lng bn mu ban
u.31/10/2011 8:58 SA 86 Nguyn Hu Tr
SYBR Green l, principeThe Polymerase Chain Reaction (PCR)
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Nguyn tc pht hin hunh quang
Molecular BeaconsNguyn tc: Dng mi c hiu vi AND ch l mt chui
oligonucleotic gn cht hunh quang R mt u v u kia cht c ch pht hunh
quang Q. Khi mi gn vo chui AND ch, R v Q tch xa nhau cho pht ra tin
hiu mu
SYBR Green I
Sondes dhybridation
Sondes TaqMan
Giai oan SA 31/10/2011 8:58keo dai
89 Giai oan annealing
GiaiNguyn keo dai oan Hu Tr
31/10/2011 8:58 SA
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Sondes TaqMan
RealTime PCR
TaqMan Procedure reporter quencher
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RealTime PCRciblelog (F2/F1)
Chun on nhanhYu cu ln ca tt c cc qu trnh ch bin thc phm l cung
cp mt sn phm khng cha vi sinh vt nguy him v cc cht c ca vi sinh vt:
Cc tc nhn gy bnh chnh trong thc phm l Listeria, Salmonella v
Campylobacter
Echantillon inconnu
n
Cc ni c t ca vi khun v cc c t nm l cc cht c ch yu cn lu tm trong
thc phm.Crossing Point (Cycles)
Courbe standard
log (F2/F1)
Mt lot cc quy trnh truyn thng, tn thi gian thng thng c p dng xc
nh , kim sot, lm gim hay loi b cc cht nhim.log (nombre de
copie)
n
Ngy nay vi s pht trin ca cng ngh sinh hc s dng cc h thng pht hin
khng th v cng ngh mu d DNA, RNA.Nguyn Hu Tr 31/10/2011 8:58 SA 94
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Chun on nhanhNhng quy trnh ny rt ngn thi gian hn, thng r hn v
khng cn i hi k nng cao s to nn mt s ci tin ln v cc tiu chun an ton
trong vic cung cp thc phm. Gn y vic s dng cc immunoassay trong cng
nghip thc phm mi pht trin nhng nhanh chng tr thnh mt cng c phn tch
c cng nhn. K thut ELISA (k thut hp th min dch lin kt vi enzyme) lm
trn mt ci a 96 ging l mt dng c s dng rng ri nht bi cc nh khoa hc
thc phm. K thut ELISA gip tit kim thi gian, nhn lc vt tri cc k thut
thng thng31/10/2011 8:58 SA 95 Nguyn Hu Tr
V d : 1/Vic pht hin Salmonella trong thc phm l mt quy trnh rt
phc tp v tn n 5 ngy nu s dng cc quy trnh tng sinh truyn thng. Bng
cch s dng cc quy trnh immunoassay th s rt ngn thi gian cn mt ngy.
2/ Cc c t nm v nm mc i hi nhiu gi tch chit, tinh sch, c c xc nh cc
chnh xc cc ha cht theo cch truyn thng. Tuy nhin, vic s dng cc ct i
lc cha cc khng th c hiu vi c t th ton b cc qu trnh tch chit, nh lng
c th tin hnh trong 30 pht.
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Chn thnh cm n
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