cac ky thuat nen CNSH 2

8/7/2019 cac ky thuat nen CNSH 2

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Chng II. Cc k thut nn

trong cng ngh sinh hc hin i

GV: Nguyn Th Kim Khang


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I. Cc k thut chnh s dng trong to DNA ti t hp(Recombinant DNA)

3. Cc vector nhn dng (cloning vector) s dng trong kthut DNA ti t hp

3.1. Khi nim

Nhn dng gene (gene cloning): qu trnh

nhn bn cc on DNA ct thnhnhiu bn ng nht v sp xp chngthnh tng dng ring bit

Vector: DNA ngn dng thng hay dngvng, DNA cn nghin cu c ci

vo DNA l

Vector nhn dng: l mt mnh nh DNA c gc ti bn,c th nhn ln trong t bo ch
http://upload.wikimedia.org/wikipedia/commons/c/c6/PGEX-3X_cloning_vector.pnghttp://upload.wikimedia.org/wikipedia/commons/c/c6/PGEX-3X_cloning_vector.png


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3.2. Yu cu ca mt vector chuyn gene

+ C im khi u sao chp (origin of replication)

+ C cc on trnh t nhn bit cho enzyme gii hn ct

+ C mt hay nhiu gene ch th: khng sinh, gene tng hp mu


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3.3. Cc ng dng ca vector chuyn gene

+ To dng, nhn dng cc on trnh t/gene to nhiu bn sao ging nhau

+ Nghin cu s biu hin ca mt on trnh/gene t

+ Chuyn gene vo t bo ca sinh vt khc

+ Sn xut cc RNA t cc DNA to dng

+ Sn xut protein tng hp t gene c to dng


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3.4. Chn la vector

+ Ph thuc vo mc ch nghin cu

+ Kiu ca t bo ch ph hp vi DNA nhn

Nhm t c hiu sut ti u cho tng mc ch nghin cu


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3.5. Cc vector nhn dng ch yuplasmids phages cosmids Cc vector khc

Phn t DNA nh, cu trc mch

vng, nm ngoi NST ca t bovk

Virus ca vi khun Vector lai nhn

to t mtplasmid vi cctrnh t cos caphage

Plasmid Ti, YAC

(yeast artificialchromosome),

Kch thc: 1.5 300 kb 2 200 kb ~ 5kb

Phn loi:+ Plasmid c s lng bn saoln (10 100)

+ Plasmid c s lng bn saonh (1 4)

+ Plasmid tip hp

+Plasmid khng tip hp

pSC101, ColE1

pBR322, pAT1153, pUC18

ng dng: vector dng nhndng 1 on DNA trong vi khun

Phn loi:+ Virulent

+ Temperate

Cc loi phages:

EMBL, GEM,phage M13, T4

ng dng: Nhndng cc on DNAc kch thc ln

Tch dng t nhnggene ln ton n hn enome

ng dng: nhn dng ccon DNA ln(40-45 kb)

ng dng: Tchdng gene, lpngn hng genome,dng trong chuyngene t bo ngvt, thc vt


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4. Cc vt ch thu nhn vector chuyn gene+ Vi khun E.coli v cc vi khun khc

+ Nm men v nm mc

+ T bo thc vt bc cao+ T bo ng vt c v


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II. Cc k thut chnh s dng trong phn tch DNA

1. Phng php tch chit DNA1.1. Nguyn tc

Ph mng t bo v mng nhn: proteinase K,

SDS (sodium dodecyl sulphate) v sarcosyl Loi b cc thnh phn khc DNA trong mu:

phenol, chloroform

Ta nucleic acid: ethanol, isopropanol


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1. Phng php tch chit DNA1.2. Tch chit DNA t m ng vt

Vt liu

Men, vi khun

Cc m ng vt (no, gan, phi, tim, thn)

Mu ng vt, cn trng


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Chn v tr mu trc khi tch gDNA

Mu Yu cu Nhit tr (oC)

S lngmu ban u

Ghi ch

M ng vt mu ti -20, -70 25-60 mg

Men G u ca

log phase

-20, -70 5x107 cells Ra bng dung

mi miVi khun 2x109 cells

Mu ng vt mu ti Proteinase K


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Homogenize

(ng nht mu)

Phase separation(Tch ri)

Centrifugation(Ly tm)

DNA precipitation(Kt ta DNA)

DNA wash(Ra DNA)

DNA solubilazation(Ho tan DNA)


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II. Cc k thut chnh s dng trong phn tch DNA1.4. Phng php tch chit RNA

ng nht mu(Homogenize)

Tch ri(Phase separation)

Kt ta RNA(RNA precipitation)

Ra RNA(RNA wash)

Ho tan RNA(RNA solubilazation)

Ph v(Disruption)


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II. Cc k thut chnh s dng trong phn tch DNA1.5. Phng php nh lng DNA, RNA

1.5.2. Phng php in di (Electrophoresis)

Nucleic acid l phn t tch in m

Phn t di chuyn t cc m n cc dng

Nguyn tc:

C 2 lai gel: Agarose gel v Polyacrylamide gel

Cc DNA khc nhau v trng lng khc nhau vin tch


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Ethidium Bromide

in di vi Agarose gel


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in di vi Agarose gel

S lng agarose gel (%) Kch thc cc on DNA

tch ri (Kb)0.6 0.8 1 2

0.9 1.2 0.5 0.7

1.2 1.5 0.2 0.5

2.0 0.1


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in di vi polyacrylamide gel

S lng acrylamide gel (%) Kch thc cc on DNAtch ri (Kb)

4 200 8005 80 200

8 40 100

11 10 - 50

Cc phn t c nh du bng ng v phng x

Pht hin v tr bng k thut phng x t ghi


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2. Phng php lai acid nucleic

2.1. Nguyn tc

-Da vo nguyn tc b sung (A-T, C-G) phn t lai

-iu kin tin hnh phn ng lainhit , ho cht (NaOH, formaldehyde) tch hai mchDNA thnh si n d dng bt cp vi nhauMu d c nh du phng x P32 pht hin v tr lai

2.2. Cc phng php lai:Southern Blot, Northern Blot, Western Blot


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Souther blot

- Nguyn l

Da vo nguyn tc b sung (A-T, C-G) ca mu d (DNA) c nh du bng ng v phng x P32


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2.2. Southern Blot- Cc bc tin hnh

DNATch DNA trnAgarose gel Chuyn DNA t gel sang mng lai

Mu dMng lai vi vchDNA c chuyn

Mu ng v phng xc vi mng lai

Cc vch DNA c th hin trn fil


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2.2. Southern Blot

- ng dng+ Nghin cu s a hnh ca DNA v nh gi s a hnhca cc on gii hn (RFLP: restriction fragment length

polymorphism)+ Xc nh trng lng phn t ca cc on gii hn vmc ging nhau gia gene NST v trnh t mu d+ C hay khng s hin din ca gene v c bao nhiu

bn sao hin din trong b genome ca sinh vt


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2.3. RFLP (Restriction fragment length polymorphism)+ L s a dng v trnh t DNA ca b genome ct DNA thnh nhngmnh nh vi cc enzyme gii hn v kch thc ca nhng mnh nyc phn tch qua chy in di (electrophoresis gel)

Nguyn tc+ Da trn s sai khc ca cc ph bng in di v di ca cc on b ctbi cc enzyme gii hn

ng dng lnh vc di truyn v chn to ging+ Cng c quan trng cho bn h genome

+ Pht hin s c mt ca gene cn tm: v tr ca cc gene mang bnh ditruyn, ri ro ca mt bnh+ Pht hin s a dng DNA qua cc c th khc nhau ca mt loi: duvn tay+ Kim tra quan h huyt thng

+ Pht hin cc th lai soma


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2.4. Northern Blot

- Nguyn l: tng t phng php lai Southern blotLai mRNA vi mu d DNA

- ng dng+ Xc nh kch thc v s lng mRNA c trong t bo

+ S khc nhau gia gene ca t bo t bin v t bo bnh thng


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1. Tch chit DNA

2. Ct DNA t cc mutocc on DNA ging nhau

3. Gn ng v phng x vou 5 DNA4. X l ho cht thnh 4 nhmring bit

5. in di sn phm trn gelpolyacrylamide

6. Chp kt qu in di bngphng x t ghi


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3. Phng php gii trnh t gene

3.2. Phng php Sanger

DNA polymerase xc tc cho vic ko di chui polynucleotide

- Nguyn l: nh vo s tng hp mch b sung cho trnh tcn xc nh nh hot ng ca enzyme DNA polymerase vcc deoxy

Deoxy l cc nucleotide mt c hai nhm OH v tr cacbons 2 & 3

Dideoxynucleotide (ddNTP): ddATP, ddCTP, ddGTP, ddTTP


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Cu trc ca cc dideoxynucleotide


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3. Phng php gii trnh t gene

3.3. Gii trnh t DNA t ng

- Nguyn l:S dng enzyme DNA polymerase v cc deoxy (phng phpSanger)


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4. K thut PCR (Polymerase chain reaction)

4.1. Khi nim

- K thut PCR: phng php nn ca CNSH hin i

- PCR (phn ng chui trng hp): on DNA c nhn ln

- Pht minh 1985 do Mullis K.- K thut c s dng trong sinh hc phn t nhn bn mton DNA trong ng nghim

DNA polymerase (t vi khun chu nhit Thermusaquaticus)

My chu k nhit Tng hp on DNA vi tc nhanh, chnh xc


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4.2. Nguyn tc

-To lng ln cc on DNA c th t DNA khun da trnc s hot ng ca DNA polymerase si mi b sung


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4.3. Thnh phn phn ng

- Nng Mg2+ v cc dNTP

- Enzyme

- Buffers (dung dch m)

- Mu DNA (Template) v mi (Primers)

- Cht khc
http://upload.wikimedia.org/wikipedia/commons/8/81/PCR_tubes.pnghttp://en.wikipedia.org/wiki/Image:Pcr_machine.jpghttp://upload.wikimedia.org/wikipedia/commons/8/81/PCR_tubes.png


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Lng enzyme: ph thuc vo mu DNA(template) v kch thcphn ng

- Enzyme

Cc c tnh ca enzyme DNA polymerase v n v khc nhau tutheo nh sn xut

Vic thay i DNA polymerase thay i hiu sut phn ng

Tnh chnh xc ca enzyme DNA polymerase ph thuc vo hottnh 35 exonuclease


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Mt s buffer cha cht hot ng b mt nh hng n ccphn ng khc

- Buffers

Hiu sut, tnh chnh xc ca enzyme DNA ph thuc vo h buffers dng

Mt s buffer nh sn mt lng ion Mg2+ nht nh lu khi ti u ho nng Mg2+


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Nng Mg2+ ti u ph thuc vo DNA polymerase, loi DNAv thnh phn buffer

-Nng Mg2+ v cc dNTPs

Nng Mg2+ cao xut hin thm cc sn phm khng c hiuNng Mg2+ thp sn phm PCR thp

dNTP l ho cht km bn chia nh lng dNTP, gim s lnlm tan v s dng li

Lng dNTP ti u l 200 M/mi loi


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- Mu DNA v mi

Nng ti u ca mu DNA:plasmid, lamda: 1pg 10 ng/50 l tng th tchmu DNA genome: 50 500 ng/50 l tng th tch

Nng mi ti u: 10 mM


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Tng DMSO trong PCR gim nhit bt mi

- Cht khc

DMSO, formamide, glycerol, betaine mu DNA ln, giu GC


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4.4. Chu trnh nhit

- Tng hp mch mi hay ko di (Extension)

- Bin tnh (Denature)

- Thc hin phn ng lai (annealing)
http://www.thuvienkhoahoc.com/tusach/images/PCR_basic_principle1.jpg


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http://www.thuvienkhoahoc.com/tusach/images/PCR_basic_principle1.jpg


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4.5. Cc yu t nh hng n phn ng PCR

- Mi v nhit nng chy ca mi

- Mu DNA lm khun: tinh sch, mu DNA khng qu di (1-1.5kb)

- Enzyme DNA polymerase

- nh hng ca cc nucleotide

- Mi trng phn ng- Thit b v dng c


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4.6. ng dng ca phng php PCR

- Tch nhanh v chnh xc tng gene

- Xc nh trnh t gene, tnh a hnh DNA, phn tch cc ch th

phn t phc v cng tc ging (cy trng, vt nui, vsv), cckiu t bin

- Chn on cc bnh di truyn, nhim trng

- Nghin cu cc mu kho c, bo tn v duy tr nhng gene qu


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5. Cc k thut xc nh tnh a hnh da trn PCR

- K thut RAPD (Randomly amplified polymorphic DNA) -a hnh cc on DNA nhn bn ngu nhin

- K thut AFLP (Amplified fragment length polymorphism) - S

a hnh chiu di ca cc phn on DNA c nhn bn

- K thut STS (Sequence tagged site) - im trnh t c nhdu

- K thut SSR ( Simple sequence repeats) a hnh trnh t lpli n gin


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6. cDNA (complementary DNA) Ngn hng cDNA

6.1. Khi nim:

- Tng hp cDNA t mRNA vi s c mt ca enzyme phin mngc (Reverse transcriptase)

-u im:-Gene thu nhn loi b c nhng trnh t khng m ho(intron).

-To dng gene m ho tng hp globine ca ngi, ng vt,

chim, protein thu tinh th mt b, ovalbumine, fibroin t tm


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6.2. K thut to cDNA

Cu trc gene


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6.2. K thut to cDNA Tch chit v tinh schRNA tng s

Gn oligo dT vo u 3

ca mRNAEnzyme sao chp ngc tng hp cDNA si kp


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Cm n rt nhiu!
http://bigteaparty.com/blog/wp-content/uploads/clone-dog-bird.jpghttp://bigteaparty.com/blog/wp-content/uploads/clone-dog-bird.jpghttp://bigteaparty.com/blog/wp-content/uploads/clone-dog-bird.jpg

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cac ky thuat nen CNSH 2