Kloning Gene

7/26/2019 Kloning Gene

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Kloning Gene

(Gene Cloning)


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What is cloning?

Isolation of (typically uncharacterized) DNA so that it

can be studied in detailsequencingdeduction of amino acid sequencegene expression studies

in vitrotranscription or translationpromoter analysisconstructs for the generation of transgenic

plants

asically! in order to fully understand the molecular

basis of many processes or traits! it is "ery helpful (if

not required) to ha"e DNA sequence information of

#ey genes$


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Cloning by gene library

%enome library

cDNA library

Cloning by PCR&'equires partial sequence information&DNA or amino acid&Wor#s ell for conser"ed genes&Wor#s ell in related species&ast

Cloning by RT-PCR

&'equires partial sequence information&amino acid or DNA&Wor#s ell hen the protein of interest is isolated

and partially sequenced


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*+' ',-*+'./ 0/

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./ 0/

./0/./ 0/

./0/

./ 0/

./0/

./ 0/

./0/./

./

0/

0/

./ 0/

./0/

./

0/./0/

denaturation (12 o+)

primer annealing (.3-43 o+)

primer extension (45 o+)

Next round6$$

./ 0/AAAAAn,,,,,n

./ 0/AAAAAn,,,,,n

first-strand cDNA synthesis by ',

./0/

,,,,,n ./0/./

,,,,,n

0/./ AAAAAn

'Nase treatment7

primer annealing (.3-43 o+)

primer extension (45 o+)

m'NA

,,,,,n0/

./

./

AAAAAn

,,,,,nAAAAAn

Next round6$$

An example of cloning based

on RT-PCR is described in

the article by Halpin et al.

(199!.


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igure 2 from 8alpin et al$ (911:)$ ,his shos

an alignment of +AD amino acid sequences

from different species$

Degenerate *+' primers ere designed

based on sequences conser"ed in most

species! and these primers ere used onmaize genomic DNA$ ,he resulting *+'

product as then used to screen a maize

cDNA library$

ull length CADcDNA sequence as obtained

"ia In"erse *+'$


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Is the identity of

the gene #non?

8as the genebeen cloned from

a different

species?

+lone "ia transposon tagging

+lone "ia ,-DNA tagging

Is there partialamino acid

sequence

a"ailable?+an the gene be

mapped?

Is transposon-

tagging possible?

Is ,-DNA tagging

possible?

Is there a mutant

in hich the gene

is deficient?

Is this a closely

related species?

;D-*+' on

genomic or cDNA

ect

Are there A+ or A+

clones a"ailable?

@ap-based cloning

An o"er"ie of gene cloning strategies


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%ene +loning


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Recombinant "#A Technology

Colonies ofE. coli$ one of the %or&horses of "#A technology.


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'estriction nzymes (


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Restriction 'nymes are )e*+ence-)pecific 'ndon+cleases That Allo%

Reprod+cible ,ragmentation of "#A

A molec+lar

scissors /ie% of

restriction

enymes.


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)ome

Commonly

0sed

Restriction'nymes

H+ndreds of

different

restriction

enymes are

a/ailable

commercially


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Restriction 'nymes )implify the Constr+ction of

Recombinant "#A


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Cloning vectors: allowing the

exogenous DNA to be inserted,stored, and manipulated mainly atDNA level.

expression vectors: allowing theexogenous DNA to be inserted,

stored, and expressed.


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1. Contains an origin of replication, allowing

for replication independent of hostsgenome.

. Contains !elective mar"ers# !election of

cells containing a plasmid

twin antibiotic resistance

blue-white screening$. Contains a multiple cloning site%&C!'

(. )asy to be isolated from the host cell.

A plasmid vector for cloning


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1. Contains an origin of replication, allowing

for replication independent of hostsgenome.

. Contains !elective mar"ers# !election of

cells containing a plasmid

twin antibiotic resistance

blue-white screening$. Contains a multiple cloning site%&C!'

(. )asy to be isolated from the host cell.

A plasmid vector for cloning


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Replica plating: transfer of the colonies from oneplate to another using absorbent pad or Velvet ( ).

transfer of colonies

+ampicillin + ampicillin+ tetracycline

these colonies have bacteriawith recombinant plasmid


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0se of ectors That Carry a )electable 2ar&er )ol/es the

#eedle in a Haystac& Problem

A /ector is a "#A that allo%s replication of the "#A fragment to be

cloned.

2odified bacterial plasmids and bacteriophage are the most common

/ectors.

The ampicillin-resistance gene allo%s selection

for cells that ha/e ta&en +p the /ector.

The lacZgene allo%s screening for /ectors that

contain a "#A insert.


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Blue white screening

Ampr

ori

p0C1

(3 &b!

MCS(Multiple cloning sites,

*ac promoter

lac+

Screening by insertional inactivation ofthe lacZ gene

he insertion of a DNA fragment interrupts the-/ of lac+ gene, resulting in non0functional geneproduct that can not digest its substrate x0gal.


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ecreated vector# blue transformantsecombinant plasmidcontaining inserted DNA#

white transformants

ecreated vector %no insert'

ecombinant plasmid %contain insert'

bac&


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4nsertion of "#A 4nto the Cloning ector

(and many other

recombinant plasmids!


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0se of ectors That Carry a

)electable 2ar&er )ol/es the

#eedle in a Haystac& Problem

The selectable mar&er is critical

beca+se only a tiny fraction of cells are

transformed (ta&e +p "#A!.


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The lacZ5ene Allo%s ,or )creening ectors 6ith An 4nsert


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Multiple cloning sites

&ultiple restriction sites enable the convenient insertion oftarget DNA into a vector

Ampr

ori

p0C1

(3 &b!

&C!%&ultiple cloning sites,

*ac promoter

lac+

7AC5AATTC5A5CTC55TACCC5555ATCCTCTA5A5TC5ACCT5CA55CAT5CA7

. T h rA s n ) er ) e r al Pro 5ly Asp Pro 8e+ 5l+ )er Thr Cys Arg His Ala )er7

'coR4 )ac4 pn4)ma4:ma4

;amH4:ba4

)al4Hinc44Acc4

Pst4 )ph4

Lac Z


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Cosmid vectors

1. tili2ing the properties of the phage cossites in a plasmid vector.

. A combination of the plasmid vector andthe C-! site which allows the targetDNA to be inserted into the head.

$. he insert can be $304 "b

I1 G i


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Gene library:a collection of different D!se"uence from an organism# each of which

has been cloned into a vector for ease ofpurification# storage and analysis.

Genomic libraries

cDNA libraries

Gene library(made from genomic DNA)

(made from cDNA- copy of mRNA

I1 Genomic

libraries

I1 G i


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$o ma%e a representative genomic libraries #genomic D! must be purified and then

bro%en randomly into fragments that arecorrect in si&e for cloning into the chosen vector.

'urification of genomic D! :

Prokaryotes


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*rea% D! into fragments randomly:

'hysical shearing:

pipeting# miing or sonicaion

Restriction en&yme digestion:

partial digestion is preferred

to get a greater lengths of D!fragments.

I1 Genomic

libraries


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cos cos

ong (left)arm

short (right)

armExogenous DNA(~20-2 !"#

, phage vector in cloning

cos cos

ong (left)arm

short (right)arm

Exogenous DNA(~20-2 !"#


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, replacementvector cloning

-. 'ac%ingwith amiture of the phagecoat proteins and

phage D!processing en&ymes

. /nfection and

formation ofpla"ues

ibrary constructed

$% &ig'tion

0%prep'r'tion o'rm 'nd genomicinserts

Gene libraries and screening


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I - cD! libraries

I-0 mR! isolation# purification

I-- 1hec% theR! integrity

I- 2ractionate and enrich mR!

I-3 4ynthesis of cD!

I-5 $reatment of cD! ends

I-6 igation to vector

Gene libraries and screening

I 2 cDNA libraries


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-.cD! libraries are very usefulfor eu%aryotic gene analysis

1ondensed protein encoded genelibraries# have much less 7un% se"uences.

cD!s have no introns genes can beepressed inE. colidirectly

!re very useful to identify new genes $issue or cell type specific (differentialexpression of genes)

cD! libraries

I 2 cDNA libraries

I 2 cDNA libraries


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I-0 mR! isolation

= 2ost e+&aryotic mR#As are polyadenylated at

their 3> ends

= oligo (dT! can be bo+nd to the poly(A! tailand +sed to reco/er the mR#A.

AAAAAAAAAAn"# ca$

I 2 cDNA libraries

I 2 cDNA libraries


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I 2 cDNA libraries

I2 cDNA libraries


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I-3 4ynthesis of cD! :

2irst stand synthesis:materials asreverse transcriptase #primer( oligo(d$) orheanucleotides) and d$'s (Fig0.0)

4econd strand synthesis:best way ofma%ing fulllength cD! is to 8tail9 the 9end of the first strand and then use acomplementary primer

to ma%e the second. (Fig-.0)

I2 cDNA libraries

I2 cDNA libraries


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?> mR#A AAAAA-3 H@-TTTTTP-?>

?>

Reverse transcriptase

Four dNTPs

AAAAA-3TTTTTP-?>

mR#A

mR#A

cDNA

cDNA

cDNA

"+plex cDNA

AAAAA-3

TTTTTP-?>

TTTTTP-?>

3>

3-CCCCCCC

Terminal transferase

dTP

Al!ali ("ydrolyaes RNA)

Purify DNA oligo(dG)

#leno$ polymerase or reverseTranscriotase Four dNTPs

?>-p5555-@H

?>

3-CCCCCCC

?>-p5555

3-CCCCCCC TTTTTP-?>

-3>

Fig%&% T"e first strand synt"esis

I2 cDNA libraries

Duple' cDNA


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?>-p55553>-CCCCCCC

H@-CC5AATTC555555

3>-55CTTAA5CCCCCC

?>-pAATTC555555

TTTTT55CTTAA5CC-@H

CC5AATTC55-3>

3>-CCCC

3>-CCCCCCC

3>-CCC

?>-p5555

?>-p5555

TTTTTp-?> -3>

TTTTTp-?>

TTTTTp-?>

-3>

-3>

TTTTT55CTTAAp-?>

H@-CC5AATTC55-3>

3>-55CTTAA5CC-@H

CC5-3>

Duple' cDNA

ingle strand-specific nuclease

#leno$ polymerase

treat $it" &coR* met"ylase

Add &colR* lin!ers

using T+ DNA ligase

&colR* digestion

,igate to vector and transfom

Fig&% econd strand synt"esis

I3 Screening procedures


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I0 4creening

$he process of identifying one particularclone containing the gene of interest fromamong the very large number of others in the

gene library .

%! &sing nucleic acid $robeto screen the library

based on hybridi'ation (ith nucleic acids!

)! Analy'e the $rotein $roduct!




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4creening libraries

ybridi&ation to identify the interested DNA orits RNA product

0. Radiolabeled probes which is complementary to a

region of the interested gene'robes:

!n oligonucleotide derived from the se"uenceof a protein product of the gene

! D! fragment;oligo from a related gene ofanother species

-. *lotting the D! or R! on a membrane

. ybridi&e the labeled probe with D!membrane(4outhern)or R! (orthern)membrane

4earching the genes of interest in a D! library




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I- 1olony and pla"ue hybridi&ation

$ransfer the D! in the pla"ue or colony to a

ylon or nitrocellulose membrane

'hage D! bind tothe membrane directly

*acterial colonies must be lysed torelease D! on the membrane

surface.

ybridi&ation (in a solution1ontaining ucleic acid probe)


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Transfer to nitrocellulose

or nylon ebrane

Denature DNA*Na+

.a/e onto ebrane

Probe (ith 0)$-labled DNA

co$leentary to

gene of interest

E1$ose to fil

2elect $ositi3e

fro aster $late

4ee$ aster

$late

2creening by $laque hybridi'ation




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/dentify the protein product of an

interested gene

0. 'rotein activity-. pression screening


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%& Pro!aryotic e'pression vector

& .aculovirus e'pression vector

/& 0ammalian e'pression vector

+& Adenoviral and retroviral vector


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Pro/aryotic e1$ression 3ector

GST-fusion

6xHis-fusion

5)T

H4)


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The en


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Kloning Gene