clonacion por recombinacion

8/13/2019 Clonacion Por Recombinacion

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Insercin de DNA en un vector

Corta y pega (Cut and paste)

(Fragmentos de PCR)

Recombinacin

(Fragmentos de PCR)


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Diana 1 Diana 2

Incorporacin de dianas

de RE por PCR

YFG


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ORF

ORFre1 re2

ORF

MCS

Pro

re1

re2

Antir

re1

re2

Antir Pro

PCR

Digestion by Re1 and Re2

+

Purification and ligation

Pro

Antir

PCR

Digestion by

restriction enzymes

Purification

Ligation

Restriction analysis

prior cloning

Transformation

3 hours

1 hour

1 hour

16 hours

16 hours

Total time = 2 days

ORF

Transformation

re2

re2

re1

re1

re1 re2

3. Restr ic t ion- independent clon ing techniques

Cloning by recombination vs traditional cloning


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Integration reaction mediated by:-Integrase (Int)

-Integration host factor (IHF)

Excision reaction mediated by:-Integrase (Int)

-Integration host factor (IHF)

-Excisionase (Xis)

Lysogenic pathway: recombination occurs between the phage circular DNA and the E. coli

chromosome via a short common sequence called "att. Att1 and att2 sites confer directionality and

specificity for recombination, so that only attB1 will react with attP1, and attB2 with attP2

3. Restriction-independent cloning techniques

Lambda phage recombination in E.coli

Landi A. (1989)Annu. Rev. Biochem. 58, 913


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Different expression systems:

E. coli, baculovirus, yeast, SFV, mammalian

cells...

Different fusions:

N- or C- terminal, MBP, GST, Trx, NusA,

GFP

attB1 ORF attB2 attP2attP1

BP clonase

pDONR

(Kanr)

entry clone

(Kanr)

LR clonase

pDEST

(Amp

r

)

expression clone

(Ampr

)

attB1 ORF attB2

attL1 ORF attL2

by product

(Kanr)

attR2attR1 suicide gene

attR2attR1

suicide gene

suicide gene

Primer 1

Primer 2

attP2attP1 suicide gene

Sistema

Gateway

PCR and purification

BP/LR reaction

Transformation

3 hours

2 hours

16 hours

Total time = 1 day


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Advantages

1) No restriction analysis of ORF prior to cloning.

2) No restriction digestion of the vector.

3) Fast, parallel sub-cloning into different expression vectors.

4) ~100% sub-cloning efficiency (no background).

5) Flexibility, automation.6) Recombination sites may serve as linkers.

Disadvantages

1) Number of available expression vectors.2) Mandatory recombination sites.

Sistema Gateway


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In Fusion / Creatortechnology (Clontech)

Cloning without restriction/ligation


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Select on Amp / Xgal plates

Vector linearization by deletion of the regioncomprised between Sal I and Hind III

Primer 5:GAAGTTATCAGTCGAC

Primer 3:ATGGTCTAGAAAGCTTIn-fusion

1rst step: PCR product cloning in donor

vector with BD In-Fusion enzyme


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Transform E. coli and select on Chloramphenicol / sucrose plates:

SacB gene inhibits sucrose metabolism. In the presence of sucrose,

bacteria that carry the SacB gene swell and die due to osmotic shock.

Any bacteria that carry non-recombinant donor Clones are eliminated

when grown on sucrose-containing media.

Creator

Cre binds to the loxP sites on both

the donor vector and the acceptor

vector, cleaves the DNA, and

covalently attaches itself to the

DNA. Then Cre catalyzes strandexchange and ligation of the DNA

so that the gene is transferred

from the donor vector into the

acceptor vector without mutation

and in the correct orientation.

Cre recombinase

Cre-loxP recombination in bacteriophage P1

2nd step: recombination in

expression vector with Cre

protein

34-bp recognition sequence

13-bp inverted repeats flanked by 8-bp spacer region

8-bp overlap regionloxPsequence

Inverted region Inverted region

Sternberg N. et al.(1981) J. Mol. Biol. 150, 467


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TOPO

DNA topoisomerase I functions both as a restriction enzyme and as a ligase.

Vaccinia virus topoisomerase I specifically recognizes the pentameric

sequence 5-(C/T)CCTT-3and forms a covalent bond with the phosphate

group of the 3

thymidine. It cleaves one DNA strand, enabling the DNA tounwind. The enzyme then religates the ends of the cleaved strand and

releases itself from the DNA. To harness the religating activity of

topoisomerase, TOPO vectors are provided linearized with topoisomerase I

covalently bound to each 3phosphate. This enables the vectors to readily

ligate DNA sequences with compatible ends.

Linearized vector with topoisomerase I

covalently bound to each 3

phosphate

Overhang invades double-

stranded DNA, displacing the

bottom strand

clonacion por recombinacion

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