Cloning & Expression Vector

b-b-b-b- The entire animal is produced from a single cell by asexual reproduction. This would allow for the creation of a human being who is genetically identical to another. Reproductive cloning Broader use of the term “cloning.” Does not create a new genetically identical individual. Research includes therapy for human mitochondria disease and others that could replace damaged or diseased tissues without the risk of rejecting another’s tissue. Could create new skin tissue for burn patients. Therapeutic cloning

What is DNA cloning? b isolation and manipulation of fragments of an organism’s genome by replicating independently as part an autonomous vector in another host species. b DNA fragment in vector will form recombinant DNA.

Basic steps in gene cloning DNA insert isolation Vector 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 in a plasmid

Cloning Considerations b b 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 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 & Vectors b Host systems: - Bacterium (E.coli) - Bacterium (E.coli) - Yeast (Saccharomyces cerevisea) - Yeast (Saccharomyces cerevisea) - Insect cells - Insect cells - Mammalian (Chinese Hamster Ovary cells) - Mammalian (Chinese Hamster Ovary cells) b 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 containing the vector from amongst those that do not (eg. antibiotic resistancy or survival under certain growth conditions. - Contain a selectable marker to distinguish host cells containing the vector from amongst those that do not (eg. antibiotic resistancy or survival under certain growth conditions.

Types of Vectors b Plasmid DNA E. coli vectors, extra-chromosomal and circular E. coli vectors, extra-chromosomal and circular b Bacteriophages Phage l – clone large DNA fragments and incorporate Phage l – clone large DNA fragments and incorporate into host genome into host genome Phage M13 – allows cloned DNA to be isolated in Phage M13 – allows cloned DNA to be isolated in single-stranded form single-stranded form b Cosmids hybrids of plasmid-bacteriophage l hybrids of plasmid-bacteriophage l b Artificial 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

viruses that infect bacteria viruses that infect bacteria known dsDNA sequence of ~ 50 kb known dsDNA sequence of ~ 50 kb linear double-stranded molecule with single-stranded complementary ends linear double-stranded molecule with single-stranded complementary ends cohesive termini (cos region) cohesive termini (cos region)

can accept large pieces of foreign DNA can accept large pieces of foreign DNA tremendous improvement over the years tremendous improvement over the years can be reconstituted in vitro can be reconstituted in vitro Desirable properties of λ 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)

b b Phage particles injects linear DNA into the cell b b DNA ligate to form circle b b Replicate to form many new phage particles which are released by cell lysis and cell death b b or DNA intergrate to host genome by site-specific recombination (lysogenic phase)

Lysis plaques of  phage on E. coli 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 apart from the chromosome of the host cell small circular dsDNA that autonomously replicates apart from the chromosome of the host cell “molecular parasites” “molecular parasites” carry one or more genes some of which confer resistance tocertain antibiotics carry one or more genes some of which confer resistance tocertain antibiotics origin of replication (ORI) --- a region of DNA that allows multiplication of the plasmid within the host origin of replication (ORI) --- a region of DNA that allows multiplication of the plasmid within the host plasmid replication: stringent or relaxed plasmid replication: stringent or relaxed

small size small size known DNA sequence known DNA sequence high copy number high copy number a selectable marker a selectable marker a second selectable gene a second selectable gene large number of unique restriction sites large number of unique restriction sites Desirable properties of plasmids:

MB 206 : Module 3 - D 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: Generic Plasmid Bacterial Replicon: Origin of replication and cis-acting control elements that regulate plasmid copy number (1-700 copies/cell) Region rich in restriction sites that make for convenient insertion of foreign DNA. Selectable Marker: Antibiotic resistance genes that facilitate the identification of bacteria which contain plasmids.

Transferring Genetic Materials Chemical method Electroporation (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 Gene The 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 Amp r Only bacteria which contain this plasmid can grow on agar containing ampicillin. All other bacteria die. Bacteri al Replico n 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

MB206 Angelia 09 32

Types of Libraries (5’UTR) (3’UTR) Angelia whole genes w/ promoters & introns (Euk.), operons (bacteria), DNA regulatory elements… Genomic Library mRNA transcript only w/ 5’ & 3’ untranslated regions (UTRs), no introns, tissue specific. cDNA Library

mRNA isolation, purificationCheck the RNA integritySynthesis of cDNATreatment of cDNA endsLigation to vector