i hc Quc Gia H Ni

Trng i hc Khoa hc T nhin

Khoa Sinh hc

THC HNH
CC K THUT C BN
TRONG CNG NGH SINH HC

Chng trnh dnh cho sinh vin ngnh Cng ngh Sinh hc
(Lu hnh ni b)

Cn b bin son: PGS. TS. Nguyn Th Vn Anh

H Ni 2015
0

Bi 1: Tch chit DNA h gen ca vi khun bng ht nano t tnh bc silica

1. Gii thiu chung
u tin, mu bnh phm c x l vi dung dch kim Mucolyse gim nht
v loi bt cc tp cht. tch chit DNA h gen ca vi khun lao (Mycobacterium
tuberculosis), mu bnh phm cha vi khun lao c x l vi dung dch m Binding
Buffer (c nng mui cao v sc cng b mt thp) v Proteinase K nhm ph v t
bo ng thi gii phng DNA h gen. Tip theo, ht nano Fe3O4 t tnh c kch thc
khong 20 nm v c bao bc bn ngoi bi mt lp silica dy khong 5 nm c tn
MagSi nano c a vo v trn u trong dung dch mu nhm to lin kt vi
DNA v d dng c thu li bi mt nam chm. Phc h DNA-MagSi nano ny c
ra vi dung dch m ra Washing Buffer 1 v Washing Buffer 2 nhm loi b
mnh xc t bo, protein... Cui cng, DNA c chit y ra khi ht MagSi nano
bng dung dch m c nng mui thp Elution Buffer. DNA tinh sch c th c
s dng cho cc ng dng tip theo nh PCR, Real time PCR pht hin gen c
hiu ca vi khun lao.

2. Ha cht v dng c
-

B kit MagPure Bacterial Nano ca hng ANABIO R&D gm cc thnh phn sau:
Thnh phn

Cat. No.

Tris HCl 50 mM pH 8

Lng
1 ng x 2 ml

Proteinase K

PK01

1 ng x 20 mg

Mag Si nano 50 mg/ml

MS01

4 ng x 1,25 ml

MB

50 ml

Resuspension Buffer

RB01

20 ml

Binding Buffer

BB01

50 ml

Washing Buffer 1

WB1-01

50 ml (m c)

Washing Buffer 2

WB2-01

100 ml

EB01

20 ml

Mucolyse Buffer

Elution Buffer
-

Cc ha cht v dng c km theo:

Ethanol tuyt i (96-100o)
ng eppendorf 1,5 ml,
Pipetman 1 ml, 200 mcl, v 20 mcl
u cn 1ml, 200 mcl, v 20 mcl v trng
Nam chm (gi t)
B n nhit,
My vortex
My soi gel c bc song UV
Bt nh du, gng tay

3. Cc bc tin hnh
3.1. Trc khi tch chit
B sung 50 ml ethanol 96-100o vo Washing Buffer 1
Ha tan Proteinase K trong 2 ml m Tris HCl 50 mM, pH 8.0
t nhit cho b n nhit 60oC
3.2. Quy trnh x l mu m
- Thm ng lng 0,2-3 ml Mucolyse buffer (MB01) vo 0,2-3 ml mu. Lc trn
u mu. nhit phng khong 10 pht tan m.
-

Ht 250 l mu x l vo ng eppendorf 1,5 ml tch chit DNA vi khun lao.

3.3.

Quy trnh x l mu dch th khc

Ht 50 l MB01 vo ng eppendorf 1,5 ml.

Thm 200 l mu vo ng eppendorf cha MB01, lc hoc pipet ln xung trn

u mu.
nhit phng khong 5 pht trc khi tch chit DNA ca vi khun lao.

3.4.

Quy trnh tch chit DNA ca vi khun lao

Thm 200 l Resuspension Buffer. Vortex 10-15 giy.

Thm 500 l Binding Buffer, 20 l Proteinase K vo mu. Vortex mu 10-15

giy. mu 60oC trong 25 pht.

Thm 200 l ethanol 96o v 50 l ht t MagSi nano vo mu. Vortex mu 10-15

giy. mu nhit phng 2-3 pht.
Ch : Vortex MagSi nano 10-15 giy m bo ht c trn u trc khi s
dng.

4 t ng ln gi nam chm tp trung ht t trong 20-30 giy hoc ti khi dch tr

nn trong. Loi b hon ton dch.
5 a ng ra khi gi nam chm, thm 600 l Washing Buffer 1. Trn u bng
cch o ng xui-ngc 10 ln hoc vortex 10-15 giy.
6 Lm theo bc 4.
7 a ng ra khi gi nam chm, thm 1 ml Washing Buffer 2. Trn u bng cch
o ng xui-ngc 10 ln hoc vortex 10-15 giy.
8 Lm theo bc 4.
Ch : cn loi b hon ton WB2 v n c th nh hng ti hiu qu tch chit
DNA.
9 Thm 50 l Elution Buffer (EB). Vortex 10-15 giy. mu 60oC trong 2-3
pht.
10 t ng ln gi nam chm tp trung ht t trong 20-30 giy hoc ti khi dch tr
nn trong. Chuyn dch cha DNA sang ng eppendorf mi.
Ch : bo qun DNA tinh sch -20oC hoc s dng ngay 5 l cho 1 phn ng
PCR/real time PCR (20-25 l) pht hin vi khun lao.

Bi 2-3: K thut cc on DNA c nhn bn ng thi

(Multiplex PCR)
1. Gii thiu chung.
K thut PCR cho php nhn bn cc on DNA hay on
gen ln nhiu triu ln thng qua hng lot chu k phn ng
tng hp ca DNA polymerase. K thut PCR yu cu phi bit
c thng tin v trnh t nucleotit hai u on DNA cn
nhn bn thit k nn cp primer c hiu.
Phn ng PCR gm 3 giai on chnh:
i) Giai on bin tnh ca DNA si kp (denaturation): giai
on ny c thc hin nhit >90 0C, thng dng l 94-950C
vi thi gian t vi chc giy n 1 pht. nhit ny DNA si
kp s b tch thnh 2 si n .
ii) Giai on gn primer (annealing of primers ): giai on
ny thng c thc hin nhit t 50-65 0C ph thuc vo
nhit tch DNA si kp thnh dng si n (Tm) ca primer.
Trong bc ny xy ra s bt cp ca primer vi cc on trnh t
b sung trn DNA. Thi gian cho giai on ny thng ch cn t
30 giy n 1 pht.
iii) Giai on ko di chui (primer extension): giai on
ny c thc hin 70-720C l nhit ti thch ca Taq DNA
polymerase. Primer c ko di theo chiu 5-3 bi Taq DNA
polymerase vi s c mt ca dNTPs di cc iu kin phn ng
thch hp. Kt qu l tng hp c cc on DNA mi b sung vi
mt phn trnh t ca DNA khun. Giai on ny thng cn
khong t 30 giy (cho cc on DNA cn nhn bn c kch thc di 1 kb) n vi pht (cho cc on DNA ln hn).
Ba giai on bin tnh, gn primer v ko di chui c
gi l mt chu k ca PCR. Sau mi chu k ta c s bn sao ca
on DNA cn nhn bn tng gp i ban u. PCR thng c
thc hin vi 25-30 chu k, v vy t mt lng DNA rt nh ban
u, sau phn ng ta c th thu c mt lng bn sao ln hn
gp nhiu triu ln so vi ban u.
K thut multiplex PCR khng khc nhiu so vi k thut
PCR thng thng. im khc bit duy nht l thay v s dng
01 cp mi nhn ln 01 loi DNA hay gen ch PCR thng
thng, multiplex PCR s dng t 3 mi tr ln nhn ln t
4

nht l 02 loi DNA hay gen ch ti cng mt thi im v cng

mt ch nhit ca chu k phn ng PCR. Cc on DNA, gen
c nhn ln ng thi thng nhm mc ch pht hin cng
mt lc nhiu i tng (loi, chng...) khc nhau hoc sng lc
cc i tng gy bnh c kh nng ng nhim mt loi mu
bnh phm (mu, nc tiu, m...). V th, multiplex PCR gip
gim thiu cc thao tc v tit kim thi gian trong chn on,
sng lc pht hin bnh.
Cc mi trong multiplex PCR cn phi c thit k sao cho Tm
phi xp x nhau c th gn b sung vo cc gen ch ti
cng mt nhit gn mi ca chu k phn ng PCR. Cc mi
trong multiplex PCR cng cn c thit k trnh hin tng t
bt cp b sung ln nhau hay bt cp khng c hiu vo
gen ch ca mi kia dn ti c ch phn ng ko di chui ca
gen kia.
Hin nay k thut multiplex PCR c p dng pht
hin cc virus ng nhim ly truyn qua ng mu nh HIV,
HBV v HCV, cc loi k sinh trng gy bnh st rt, cc loi vi
khun v nm gy bnh ph khoa, cc vi khun gy bnh ng h
hp, gy bnh ng rut, ng c thc phm...
Trong th nghim ny sinh vin s dng cc primer c thit
k v k thut multiplex PCR pht hin ng thi gen c hiu
cho vi khun lao v gen ni chun b-globin trong mu bnh phm nghi nhim lao. Quy
trnh th nghim ny l mt phn kt qu ca nhm nghin cu
thuc phng Sinh hc Nano v ng dng, Phng Th nghim Trng im cng ngh
Enzym v Protein.

2. Ho cht.
Taq DNA polymerase,
m cho Taq DNA polymerase c cung cp nng gp 10
ln nng hot ng (10xTaq buffer),
dNTPs nng 25 mM cho mi loi dNTP,
Primers (trong th nghim ny l IS-6110 Fw, IS-6110 Rv, Globin
Fw, Globin Rv.
DNA genome ca MTB (chun dng)
Plasmid pGEM cha gen ni chun -globin (ni chun)
DNA khun nng 5-15 ng/l c tch chit t bi 1.
dH2O kh trng
m TE
5

3.Dng c v my mc:
- Pipetman (1000, 100, 20l),
- u cn vng, u cn xanh, ng eppendorf 0,5 ml,
- Gi ng eppendorf,
- My PCR, my in di ngang, b ngun in di, v my soi UV

4. Cch lm:
Chun b mt hn hp ca cc thnh phn chung nhau cho 5
phn ng:
trong , cc mu khc nhau s khc nhau v DNA khun, ngoi
ra cn c 1 mu kim tra khng c DNA khun. Th tch cui
cng ca mi phn ng l 25 l, nng cui cng ca primer l
1,5 ng/l, dNTPs l 0,2 mM, DNA khun l 0,5-0,7 ng/l
Chun b mt ng eppendorf, cho vo :
a) 10 l m Taq (10xTaq buffer)
b) 5 l IS6110 Fw (25 ng/l)
c) 5 l IS6110 Rv (25 ng/l)
d) 5 l Globin Fw (25 ng/l)
e) 5 l Globin Rv (25 ng/l)
f) 1 l dNTP 20 mM
g) 2 l Taq (5 nv/l)
h) 5 l plasmid ni chun (5 ng/l)
i) 42-45 l dH2O
Tng th tch cui cng l 80-83 l.
Chia u hn hp cho 4 ng eppendorf nh du t 1-4,
mi ng 20 l.
Sau b sung ln lt vo cc ng nh sau:
ng
ng
ng
ng

#1: 5 l m TE (C -)
#2: 5 l DNA ca chng dng MTB (C +)
#3: 5 l DNA tch t mu bnh phm 1
#4: 5 l DNA tch t mu bnh phm 2

Trn hn hp phn ng nh nhng bng pipetman, trnh to

bt. Nu c bt th cn ly tm ngn (khong 10.000v/p, 1
pht) loi bt v cho ton b dch phn ng tp trung
xung y ng nghim.
Tin hnh t multiplex-PCR cho 30 chu k vi ch nhit
nh sau:
Bc lm bin tnh 94 oC: 30 giy
Bc gn mi 60 oC: 30 giy
Bc ko di chui 72 oC: 30 giy

t b sung bc gi 72oC: 7 pht v gi mu n khi

phn tch 4 oC
Sau khi chy PCR xong, tin hnh chy in di cc mu trn
gel agarose 2% trong m 1X TAE thu nhn cc bng DNA
nhn bn. Phn tch bng thu c so vi mu DNA i chng
xc nh mu c nhim bnh hay khng.