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8/11/2019 Ingles Sobre Clonacion

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Cloning in bacteriaPresenter: Vito Baraka

(BSc,MSc Cand.)


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Introduction

DNA cloning involves separating a specific gene or DNA

segment from a larger chromosome, attaching it to a smallmolecule of carrier DNA, and then replicating this modifiedDNAboth an increase in cell number and the creation of multiplecopies of the cloned DNA in each cell. A cloneis an identicalcopy

First proposed by Peter Lobbane et al 1973 at the StanfordUniversity.Lobbanet al1973 "Biochemical Method for Inserting New Genetic Information intoDNA of Simian Virus 40: Circular SV40 DNA Molecules Containing Lambda PhageGenes and the Galactose Operon of Escherichia coli

Cloning was made possible by the discovery, isolation andapplication of restriction endonucleases by Werner et al(1970)


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Overview cloning bacteria

Key steps

1. Cutting DNA at preciselocations.

2. Cloning vector3. Joining two DNAfragments covalently

4. Transformation to a hostcell

5. Selecting or identifyinghost cells that containrecombinant DNA


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1.How can we cut DNA at specific sites?

Restriction endonuclease enzymestype II binds to DNA at a specific

sequence and make a double-stranded cut at or near thatsequence

Can form sticky or cohesive ends orBlunt ends

Most recognition sequences arepalindromic

eg

..live not on evil

The sticky ends are importantfor annealing. DNA ligase willeventually form a covalentbond btn the sugar-

phosphate


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2.Cloning vectors

Allow amplification of inserted DNA fragments

Developed fromnaturally occurring bacterialplasmids

Contain an origin of replication(ori )

Contain numerous restriction

sites

Contain genes that conferresistance to antibiotics, thusallowing selection of

bacterial colonies carrying theplasmid

Introduced into competentbacterialcells by transformation


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2.Cloning vectors cont..Expression vectors

Vectors that can yield the proteinproducts of the cloned genes.

For active gene expression:

i)strong promoter

ii)ribosome binding site near ATG

codon

The main function of an expressionvector is to yield the product of agene, therefore a strong promoter is

necessary.

The more mRNA is produced, themore protein product is made.

Oligohistidine regions likethis have a high affinity formetals like nickel, so

proteins that have suchregions can be purifiedusing nickel affinitychromatography

ATG

(His)6

MCS


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3. Joining two DNA fragments covalently

DNA LigaseDNA ligase ,much more efficient with

sticky ends, for blunt end not efficient.

Linkers: short, double-stranded, blunt-ended, DNA fragments.Contain a stickyend restriction site.

Adaptors.These are linkers with cohesiveends or a linker digested with RE,before ligation. By adding adaptors tothe ends of a DNA, sequences that areblunt can be converted into cohesiveends.

Homopolymer tailing (TdT). Terminaldeoxynucleotidyl transferase addssingle stranded tail in the presence ofone nucleotide producing sticky ends.

Fig1.Linkers

Fig 2.Adaptor

3 Li ti t


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3.Ligation cont..

What next ???


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4.Transformation a host cell

Most bacteria, including E. coli,only take up a limited amountof DNA.In GE bacteria aretreated to increase uptake.

Following treatment, cells aresaid to be competent

Treating growingE. coli

cells withsolutions (CaCl2 and MgCl2)the cells are madecompetent to take up DNA.

Incubate thawed cells with DNA,then heat-shock at 42C for30 seconds (DNA is taken upby cells).

Spread plate out on appropriateselection media

1: Mix competentcells and plasmidvector

2: Keep on icefor 30 min

3: Heat shock at42C 30 sec

4:Plate out cells on

Ampicillin

selective medium


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4.Transformation contOnly tranformed

E.Coli will grow onAmplicilinsupplemented media

Transformantionefficiency:

Can be calculated as

number of coloniesformed per mg of inputDNA.


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5:Selecting/Screening

Transformed cellswill grow

on selective media

Others will not!


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5.Selection/screening cont

Interrupting LacZ

Lac Z gene

Beta-galactosidase

X-gal

(colorless)Gal + X(Blue dye)blue colonies

pUC18


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Analysis of recombinants

For analysis ofrecombinants:

Colony PCR and resolve on

agarose

Perform restriction digestionanalysis

Sequencing

Immunoblotting forexpression vector


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Some cloning applications

Human insulin, Human growth hormone,

interferons, growth factors, blood clotting factors,Plasminogen activator, tumor necrosis factor,novel recombinant antibodies

Synthetic peptides as recombinant vaccines(Hep B, malaria, rabies, HIV/AIDS)

Gene Transfer in plants---increased yield,improved tolerance, herbicide res., diseaseresistance


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Aknowledgment

Steven and Danielle

Classmates

All IPMB staff


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...je vous remercie de votre attention

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