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Bi ging k thutxc nh trnh t gen


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K thut xc nh trnh t genK thut xc nh trnh t gen Cng ngh sinh hc nanoCng ngh sinh hc nano

Ni dungNi dung


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K thut xc nh trnhK thut xc nh trnh

t gent gen


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Ni dungNi dung

Nguyn l ca cc phng php phn tch trnh t genNguyn l ca cc phng php phn tch trnh t gen

Phng php SangerPhng php Sanger

Phng php MaxamPhng php Maxam--GilbertGilbert

Phng php phn tch trnh t gen t ngPhng php phn tch trnh t gen t ng

Ho hc/ Phn ng chui (dy chuyn) polymeraseHo hc/ Phn ng chui (dy chuyn) polymerase

Thit bThit b

Cc phn mm/ Cc c s d liu gen quc tCc phn mm/ Cc c s d liu gen quc t

X l s liu/ ng k d liu genX l s liu/ ng k d liu gen


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DNA/RNADNA/RNA


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Cu trc phn t ca ng Ribose v DeoxyriboseCu trc phn t ca ng Ribose v Deoxyribose


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Trng thi t nhin Trng thi bin tnh si n Trng thi li tnh


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INHNGHA: GII TRNH T GENINHNGHA: GII TRNH T GEN

L phng php thc nghim xc nh trnh tnucleotide ca mt on ADN Xc nh chnh xc trt t sp xp ca cc cp trngmt on ADNwww.accessexcellence.org/AE/AEPC/NIH/gene27.html Phn tch trnh t ca cc n v mang thng tin ditruyn.

www.mwgbiotech.com/html/glossary/glossary_overview.shtml


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Lch s phn tch trnh t genLch s phn tch trnh t gen 1870 Mischer:Pht minh DNA1870 Mischer:Pht minh DNA 1940 Avery: DNA l cht liu di truyn1940 Avery: DNA l cht liu di truyn 1953 Watson & Crick: Cu trc xon kp ca DNA1953 Watson & Crick: Cu trc xon kp ca DNA 1965 Holley: Trnh t ca tRNA nm men1965 Holley: Trnh t ca tRNA nm men 1977 Wu: Trnh t ca u dnh DNA thc khun1977 Wu: Trnh t ca u dnh DNA thc khun 1977 Sanger:Phng php dng chui bng1977 Sanger:Phng php dng chui bng

dideoxydideoxy Maxam & Gilbert:Phng php phn giiMaxam & Gilbert:Phng php phn giiho hcho hc

1980 Messing: Chn dng trong thc khun M131980 Messing: Chn dng trong thc khun M13 1986 Hood et al: T ng ho mt phn1986 Hood et al: T ng ho mt phn 1990 Phn tch trnh t bng chu trnh nhit (PCR);1990 Phn tch trnh t bng chu trnh nhit (PCR);

cc enzyme c hon thin c trnh t; cccc enzyme c hon thin c trnh t; cch thng d hunh quang c hon thin (ch thng d hunh quang c hon thin (cqua ng mao dn)qua ng mao dn)

2002 T ng ho phng th nghim2002 T ng ho phng th nghim

115

1501.500

25.000

50.000

200.000

Hiu sutHiu sutbp/ngi/nmbp/ngi/nm

53.000.000


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Phn b s lng genom c gii trnh tPhn b s lng genom c gii trnh thon ton cc sinh vthon ton cc sinh vt

Archea (16)Archea (16)

Eukarya (20)Eukarya (20)

Bacteria (139)Bacteria (139)

Viruses (1500)Viruses (1500)


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NGUYNL CA CC PHNG PHPNGUYNL CA CC PHNG PHPPHN TCH TRNH T GENPHN TCH TRNH T GEN

Phng php SangerPhng php Sanger

Frederick (Fred) Sanger


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Phng php MaxamPhng php Maxam--GilbertGilbert

Walter GilbertWalter Gilbert


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Phn ng chui/dy chuyn (ca/bng/nh/do)Phn ng chui/dy chuyn (ca/bng/nh/do)PolymerasePolymerase

Polymerase Chain Reacti

on (

PCR)

Polymerase Chain Reacti

on (

PCR)

Kary Mullis

Gii Nobel v ho hc, 1993


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http://www.nobel.se/chemistry/laureates/1993/mullis-autobio.html

Mullis, K.B. (1990) The unusual origin of the polymerase chain reaction.Scientific American. 262 (4) 56-65.

devised by Kary Mullis c1983

POLYMERASE CHAIN REACTION - PCR

A 'licence' to do molecular biology

A key central technique that has revolutionised molecular andconsequently cell biology


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Schematic illustration ofPCR stepsSchematic illustration ofPCR stepsS minh ha cc buc PCRS minh ha cc buc PCR

From:From: Recombinant DNARecombinant DNA by Watson,by Watson,Gilman, Witkowski & ZollerGilman, Witkowski & Zoller


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Target AmplificationTarget Amplification

No. of No. Amplicon

Cycles Copies of Target

1 2

2 4

3 8

4 16

5 32

6 64

20 1,048,576

30 1,073,741,824

1 cycle = 2 Amplicon








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Cc phng php xc nhCc phng php xc nh

trnh t genetrnh t gene1. Phng php Maxam-Gilbert (ho hc)

Phng php c pht minh u tin nhng nnay t c s dng, ch dng chofootprinting

2. Phng php Sanger (enzyme)S dng DNA polymerase, primer, dNTP v mt

lng nh ddNTP ( kt thc chui)


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5-

Xl bng hocht ct chiu ti mtbase no

4 ng

G-rxn A>G-rxn

T>C-rxnC-rxn

5-

5-

5-

C

Khi phn ng kt thc mi si chc ct 1 ln

ddNTP Reaction Mix

PHNG PHP MAXAM-GILBERTSi nh du (P32)

C

C

Trnh t c c trc tip

autoradiograph of dried

sequencing gel

C T A G

3GC

ATTCCAT

AGG5


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5-

DNAPolymerase

+dNTPs

+ one of:

primer

4 tubes

ddCTPddTTP ddATP

ddGTP

5-

5-

5-

C

C

C

In the ddGTP reaction there is arandom chance a ddGTP will be

incorporated across from a C

C T A G

ddNTP Reaction Mix

5CG

TAAGGTAT

CC3

PHNG PHPPHNG PHP SANGERSANGEREnd labeled

Sequence is complementary

to the strand being sequenced!

autoradiograph of dried

sequencing gel


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DNA Sequencing sau khi to dng phn tDNA Sequencing sau khi to dng phn t

Plasmid (or phage)with cloned DNA

fragment

Primer Binding sites

Clonedfragment

Primer


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DideoxyDideoxy--NucleotidesNucleotides

O(P)-(P)-(P)-

H

Base

dNTP ddNTP

O(P)-(P)-(P)-

H H

Base

If a ddNTP is added toa growing DNA chain,the sequence isterminated, becauseanother base can not

be added

OH


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H

P

O

OH

OH

HO

O

O

CH2

NH2

N

N N

N

Sugar

Base

Phosphate

3

5

2

14

DideoxynucleotidesDideoxynucleotides DNA Sequencing using theDNA Sequencing using the

Sanger metho

d invo

lves theSanger metho

d invo

lves theuse of23use of23--dideoxynucleotidedideoxynucleotidetriphosphates in addition totriphosphates in addition toregular 2regular 2--deoxynucleotidedeoxynucleotidetriphosphatestriphosphates

Because 23Because 23--dideoxynucleotidedideoxynucleotidetriphosphates lack a 3triphosphates lack a 3hydroxyl group, and DNAhydroxyl group, and DNApolymerization occurs only inpolymerization occurs only inthe 3 direction, once 23the 3 direction, once 23--

dideoxynucleotidedideoxynucleotidetriphosphates aretriphosphates areincorporated, primer extensionincorporated, primer extensionstopsstops

H

23-dideoxynucleotidemonophosphate

2-dideoxynucleotidemonophosphate


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H

P

O

HO

O

O

CH2

HOH

P

O

O

HO

O

O

CH2

H

P

O

OH

HO

O

O

CH2

NH2

N

N

N

N

O

O

NH2N

NH

N

N

N O

NH2

N

2323dideoxydideoxy--

nucleotidesnucleotidesTerminateTerminateDNADNA

ReplicatonReplicatonOH

P

O

HO

O

O

CH2

HO

H

H OH

P

O

OH

O

O

CH2

OH

H

P HO

O

O

CH2

H

O

H2O


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Phng php phn tch trnh t gen tPhng php phn tch trnh t gen tngng

Ho hc:Phn ng chui (dy chuyn) polymeraseHo hc:Phn ng chui (dy chuyn) polymerase

Cc thit bCc thit b

Cc phn mm/ Cc c s d liu gen quc tCc phn mm/ Cc c s d liu gen quc t X l s liu/ ng k d liu genX l s liu/ ng k d liu gen


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The dideoxy method has beenmodified so it can be done in onetube. In this case all of theddNTPs are labeled with different,coloured fluorescent molecules

This makes automation muchsimpler, reduces the cost of the

reactions, and speeds up theprocess tremendously. Largesequencing centres use thismethod and can read millions ofbp every single day


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Sequencing DataSequencing Data


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My gii trnh t DNA t ngMy gii trnh t DNA t ng(Automated DNA Sequencers)(Automated DNA Sequencers)

ABI:ABI 377, 310, 3100, 3100 Avant, 3700, 3730ABI:ABI 377, 310, 3100, 3100 Avant, 3700, 3730

Amersham: MegaBace 500, 100Amersham: MegaBace 500, 100

BeckmanBeckman--Coulter: CE2000, 8000Coulter: CE2000, 8000

LiLi--Cor:Cor:

Shimatzu:DSQ1000, 2000Shimatzu:DSQ1000, 2000

Ti Vit Nam:Ti Vit Nam:

ABI 3100 Avant (4 capillaries): 5/ VCNSH, TTCNSH (HQGHN); HYHN,ABI 3100 Avant (4 capillaries): 5/ VCNSH, TTCNSH (HQGHN); HYHN,VVSDTTW (sp mua 2?)VVSDTTW (sp mua 2?)

ABI 310 (1 Capillary): 4/H Thi Nguyn; VDTNN, VKHHS, H CnABI 310 (1 Capillary): 4/H Thi Nguyn; VDTNN, VKHHS, H Cn

Th?Th? ABI 377: 1/ VKHHSABI 377: 1/ VKHHS

Pharmacia Biotech ALF: 4/HKHTN HQGHN, HKHTN HQGtpHCM,Pharmacia Biotech ALF: 4/HKHTN HQGHN, HKHTN HQGtpHCM,VCNSH, HBKVCNSH, HBK

BeckmanBeckman-- Coulter CE2000: 2/Vin Pasteur TPHCM; TT Welcome Trust)Coulter CE2000: 2/Vin Pasteur TPHCM; TT Welcome Trust)

??? Vin Chn nui, VQY108,H

VQY...??? Vin Chn nui, VQY108,H

VQY...


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ABI sequencersABI sequencers

ABI 377

ABI 373ABI 371


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ABI Automated SequencersABI Automated Sequencers(contd.)(contd.)

ABI 310ABI 3700

ABI 3100AvantABI 3100


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Lgii trnh t DNALgii trnh t DNA

123123123

654654654

Khung c m 2Khung c m 2Khung c m 1Khung c m 1


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Bng n thng tin sinh hc !!!

Cc C s d liu gen Quc tCc C s d liu gen Quc tNCBI (USA); EMBL/EBI (EU); DDBJ (Nht Bn)NCBI (USA); EMBL/EBI (EU); DDBJ (Nht Bn)


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NCBINCBI-- Trung tm Quc gia v Tin hc Cng nghTrung tm Quc gia v Tin hc Cng nghSinh hc Hoa KSinh hc Hoa K

C s d liu gen: GENBANKGENBANK


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S trnh t gen c ng k trong cc Ngn hng genS trnh t gen c ng k trong cc Ngn hng genquc t tng ln hng nm theo hm s m (exponential)quc t tng ln hng nm theo hm s m (exponential)

0

200.000

400.000

600.000

800.000

1.000.000

1.200.000

1965 1970 1975 1980 1985 1990 1995

Currently5.4 MillionSequenceEntries in

GenBank

4.6 Billion Bases

from ~50,000Species


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