Cloning & Expression Cloning & Expression VectorVector

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What is DNA cloning?What is DNA cloning?

isolation and manipulation of isolation and manipulation of fragments of an organism’s genome fragments of an organism’s genome by replicating independently as part by replicating independently as part an autonomous vector in another host an autonomous vector in another host species. species.

DNA fragment in vector will form DNA fragment in vector will form recombinant DNA.recombinant DNA.

Basic steps in gene cloningBasic steps in gene cloning

DNA

insert

isolationVector

restriction

ligation

Recombinant DNA

Transformation/amplification

Host cells

Selection / identification of clones

Validation of clones –analyses RE, Southern blot, PCR, DNA sequencing

Positive recombinant DNA

The cloning of DNA The cloning of DNA in a plasmidin a plasmid

Cloning ConsiderationsCloning Considerations

Choosing a vector• Plasmids limited to small molecules• Bacteriophages (phages) are viruses that

infect bacteria• Phages can carry DNA inserts up to

15,000 nucleotides long

Cloning ConsiderationsCloning Considerations

Choosing a host• Bacteria very good, but limitations• Bacteria lack ability to modify proteins and limited

size of insert• Yeast (Saccharomyces cere isiae) excellent for

many applications• Occasionally necessary to clone genes into

specific animal or plant hosts – more difficult but possible

Hosts & VectorsHosts & Vectors

Host systems: Host systems: - Bacterium (- Bacterium (E.coliE.coli)) - Yeast (- Yeast (Saccharomyces cereviseaSaccharomyces cerevisea)) - Insect cells- Insect cells - Mammalian (Chinese Hamster Ovary cells)- Mammalian (Chinese Hamster Ovary cells)

Cloning vectors Cloning vectors - derived from natural replicons- derived from natural replicons - Capable of replicating and isolation from host.- Capable of replicating and isolation from host. - Contain a selectable marker to distinguish host cells - Contain a selectable marker to distinguish host cells

containing the vector from amongst those that do not (eg. containing the vector from amongst those that do not (eg. antibiotic resistancy or survival under certain growth antibiotic resistancy or survival under certain growth conditions.conditions.

Types of VectorsTypes of Vectors Plasmid DNAPlasmid DNA E. coliE. coli vectors, extra-chromosomal and circular vectors, extra-chromosomal and circular Bacteriophages Bacteriophages Phage l – clone large DNA fragments and incorporate Phage l – clone large DNA fragments and incorporate into host genomeinto host genome Phage M13 – allows cloned DNA to be isolated in Phage M13 – allows cloned DNA to be isolated in single-stranded formsingle-stranded form CosmidsCosmids hybrids of plasmid-bacteriophage lhybrids of plasmid-bacteriophage l Artificial chromosomesArtificial chromosomes - Cloning of very large genomic fragments - Cloning of very large genomic fragments - BACs (bacterial artificial chromosomes)- BACs (bacterial artificial chromosomes) - YACs (yeast artificial chromosomes- YACs (yeast artificial chromosomes

http://dwb.unl.edu/Teacher/NSF/C08/C08Links/mbclserver.rutgers.edu/~sofer/lambdaMap.gif

• viruses that infect bacteriaviruses that infect bacteria

• known dsDNA sequence of ~ 50 kbknown dsDNA sequence of ~ 50 kb

• linear double-stranded molecule with linear double-stranded molecule with

single-stranded complementary endssingle-stranded complementary ends

• cohesive termini (cos region)cohesive termini (cos region)

• can accept large pieces of foreign DNAcan accept large pieces of foreign DNA

• tremendous improvement over the tremendous improvement over the

yearsyears

• can be reconstituted can be reconstituted in vitroin vitro

Desirable properties of Desirable properties of λλ

phage:phage:

Bacteriophage

phage genome - linear 48.5 kb genome.

Each ends consists of cos (cohesive) sites – 12 bp cos ends Cos ends allows DNA circularization in the cell

Central region of genome are non-essential portions and can be replaced by foreign DNA (up to 23kb)

Phage particles injects linear DNA into the cell

DNA ligate to form circle

Replicate to form many new phage particles which are released by cell lysis and cell death

or DNA intergrate to host genome by site-specific recombination (lysogenic phase)

Lysis plaques of Lysis plaques of phage onphage on E. coliE. coli bacteria. bacteria.

bacteriophage

plaques

bacteria lawn

Plaques: the clear areas within the lawn where lysis and re-infection have prevented the cells from growing.

• small circular dsDNA that autonomously replicates small circular dsDNA that autonomously replicates

apart apart from the chromosome of the host cellfrom the chromosome of the host cell

• ““molecular parasites”molecular parasites”

• carry one or more genes some of which confer carry one or more genes some of which confer

resistance toresistance tocertain antibioticscertain antibiotics

• origin of replication (ORI) --- a region of DNA that origin of replication (ORI) --- a region of DNA that

allows multiplication of the plasmid within the allows multiplication of the plasmid within the

hosthost

• plasmid replication: stringent or relaxedplasmid replication: stringent or relaxed

small sizesmall size

known DNA sequenceknown DNA sequence

high copy numberhigh copy number

a selectable markera selectable marker

a second selectable genea second selectable gene

large number of unique restriction siteslarge number of unique restriction sites

Desirable properties of plasmids:Desirable properties of plasmids:

http://www-micro.msb.le.ac.uk/109/GeneticEngineering1.gif

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Transferring of Genetic Materials

Bacterial transformation Process by which bacterial cells take

up naked DNA molecules.

If the foreign DNA has an origin of replication recognized by the host cell DNA polymerases, the bacteria will replicate the foreign DNA along with their own DNA.

Polycloning Site:GenericPlasmid

Bacterial Replicon:

Origin of replication and cis-acting control elements that regulate plasmidcopy number (1-700 copies/cell)

Region rich in restriction sitesthat make for convenientinsertion of foreign DNA.

Selectable Marker:Antibiotic resistance genesthat facilitate theidentification of bacteriawhich contain plasmids.

Transferring Genetic Materials

(use salt and heat-shock) to promote DNA uptake.

electric shock to facilitate DNA uptake.

Method of transferring genetic material to recipient cells without the need for conjugation.

Advantage vs Disadvantage

•Heat-Shock Transformation:

– Quick and dirty but not very efficient

•Electroporation:

– More tedious but also far more efficient.

-Galactosidase GeneThe protein encoded by this gene turns cells blue. Insertion of foreign DNA in the middle of this gene screws up the protein so cells appear white. PCS

AmprOnly bacteria which contain this plasmid can grow on agar containing ampicillin. All other bacteria die.

Bacterial Replicon

In the presence of selective media and X-Gal only transformed cells grow and those without inserts turn blue.

CLONING VECTORS Cloning vectors are DNA molecules that are used to

"transport" cloned sequences between biological hosts and the test tube.

Cloning vectors share four common properties:

1. Ability to promote autonomous replication.

2. Contain a genetic marker (usually dominant) for selection.

3. Unique restriction sites to facilitate cloning of insert DNA.

4. Minimum amount of nonessential DNA to optimize cloning.

PLASMID VECTORS

Plasmid vectors are ≈1.2–3kb and contain:

replication origin (ORI) sequence

a gene that permits selection,

Here the selective gene is ampr; it encodes the enzyme b-lactamase, which inactivates ampicillin.

Exogenous DNA can be inserted into the bracketed region .

SELECTIVE MARKER

Selective marker is required for maintenance of plasmid in the cell.

Because of the presence of the selective marker the plasmid becomes useful for the cell.

Under the selective conditions, only cells that contain plasmids with selectable marker can survive

Genes that confer resistance to various antibiotics are used.

Genes that make cells resistant to ampicillin, neomycin, or chloramphenicol are used

ORIGIN OF REPLICATION

Origin of replication is a DNA segment recognized by the cellular DNA-replication enzymes.

Without replication origin, DNA cannot be replicated in the cell.

MULTIPLE CLONING SITE Many cloning vectors contain a

multiple cloning site or polylinker: a DNA segment with several unique sites for restriction endo- nucleases located next to each other

Restriction sites of the polylinker are not present anywhere else in the plasmid.

Cutting plasmids with one of the restriction enzymes that recognize a site in the polylinker does not disrupt any of the essential features of the vector

Restriction mapping: determining the order of restriction sites in a cloned fragment:

Gel electrophoresis: separates DNA fragments by molecular weight

Southern Blot analysis: DNA is transferred ("blotted") to filter paper.Filter is exposed to a DNA probe. Binds specifically to target DNA immobilized on filter

DNA sequencing: provides complete order of bases in a DNA fragment       

RECOMBINANT DNA R.E. are a useful tool for analysing Recombinant DNA ▪ checking the size of the insert ▪ checking the orientation of the insert ▪ determining pattern of restriction sites within insert Sometimes it is important to determine the orientation

of the DNA insert in relation to the vector sequence. This can be done simply by restriction digest using

enzyme(s) which cut the vector sequence near to the insert and cut within the insert sequence (asymmetrically).

APPLICATIONS Cloning DNA fragments Generating Libraries: essential step

for genome mapping Positional cloning – discovering

disease genes Discovering genes from e.g. Protein

sequence

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Types of Libraries

(5’UTR) (3’UTR)

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