Cloning

RECOMBINANT DNA TECHNOLOGY

What is Genetic What is Genetic Engineering?Engineering?

• Genetic engineering (GE) is the transfer of genes from one organism to another through means that do not occur in nature, but through human intervention. This involves isolating and then moving genes within and without different species by recombinant DNA techniques and other manipulation of the genetic construct outside the traditional practices such as sexual and asexual breeding, hybridization, fermentation, in-vitro fertilization and tissue culture.

The Term of Gene The Term of Gene ManipulationManipulation

Gene Manipulation is defined as the formation of new combinations of heritable material by the insertion of nucleic acid molecules ， produced by whatever means outside the cell, into any virus, bacterial plasmid or other vector system so as to allow their incorporation into a host organism in which they do not naturally occur but in which they are capable of continued propagation. Also named gene cloning.

•Gene cloning is production of many identical copies of the same gene.

•If the inserted gene is replicated and expressed, we can recover the cloned gene or protein product.•Cloned genes have many research purposes: determining the base sequence between normal and mutated genes, altering the phenotype, obtaining the protein coded by a specific gene, etc. •Humans can be treated with gene therapy: alteration of the phenotype in a beneficial way

DNA Manipulation

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• Recombinant DNA (rDNA) contains DNA from two or more different sources– Requires:

• A vector– introduces rDNA into host cell– Plasmids (small accessory rings of DNA from bacteria) are

common vectors– Phage vectors (bacterial viruses) can also be used

• Two enzymes to introduce foreign DNA into vector DNA– A restriction enzyme - cleaves DNA

– Bacterial enzyme that stops viral reproduction by cleaving viral DNA

– Act as molecular scisssors (cut plasmids and foreign human DNA)

– Produce short single stranded “sticky ends” where insertions of foreign DNA can be made

– A DNA ligase enzyme - seals DNA into an opening created by the restriction enzyme

DNA ManipulationDNA Manipulation

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Cloning a Cloning a Human GeneHuman Gene

Restriction enzyme EcoRI–Bacterial enzyme that stops viral reproduction by cleaving viral DNA

–Act as molecular scisssors (cut plasmids and foreign human DNA)

–Produce short single stranded “sticky ends” where insertions of foreign DNA can be made

What is a Gene? a What is a Gene? a Genome?Genome?

• A gene is a unit of inheritance. For example, children tend to look like their parents. We inherit our features through our genes, half of which come from one parent, and half from the other.

• A gene is also defined by a digital code of just four DNA bases (A,T,G & C) that is nearly universal for all known life forms, whether viral, bacterial, fungal, plant, animal or human. The average size gene of bacteria is about 1,000 bases long.

• Since genes are encoded by the DNA bases that comprise the linear strands of a chromos-ome, the genes are arranged in linear order along chrom-osomes, and they can be mapped.

Why Clone DNA?Why Clone DNA?• A particular gene can be isolated and its

nucleotide sequence determined• Control sequences of DNA can be

identified & analyzed• Protein/enzyme/RNA function can be

investigated• Mutations can be identified, e.g. gene

defects related to specific diseases• Organisms can be ‘engineered’ for

specific purposes, e.g. insulin production, insect resistance, etc.

How is DNA cloned?How is DNA cloned?

Cell-based DNA cloning Cell-free DNA cloning (PCR)

Clone in dividing cellsClone in dividing cells

Clone Clone ininPPCCRR

Restriction Endonucleases--The Molecular Scissors

Host enzymes that prevent the invasion of foreign DNAs such as viral DNA, by cutting them up.

These enzymes cut within the foreign DNAs, rather than chewing them away from the ends.

Restriction

Endonucleases

These enzymes recognize a specific DNA sequence (4-12bp) which is twofold symmetry and cut both DNA strands

Some enzymes make staggered cuts GAATTCCTTAAG

Some make even cuts CCCGGGGGGCCC

Sticky end

Sticky end

DNA ligase covalently links two DNA strands

Restrictionenzyme

Restrictionenzyme

Ligase

Ligase

5’ 3’

5’3’

Plasmid: a cloning vector or Plasmid: a cloning vector or vehiclevehicle

•Restriction Enzyme Mechanisms:• Preparation of DNAs to be joined• (a)Staggered cut: leaves “sticky

ends”

•Restriction Enzyme Mechanisms: Preparation of DNAs to be joined:

• (b) Blunt End

Ligation of DNA cut with a Ligation of DNA cut with a Restriction EnzymeRestriction Enzyme

•Staggered “sticky ends”

Ligation of DNA cut with a Ligation of DNA cut with a Restriction EnzymeRestriction Enzyme

•Role of T4 DNA Ligase

Selectable Markers:Selectable Markers:

cDNA LibrarycDNA Library•Used to obtain

functional eukaryotic coding regions.

•E. coli does not process introns.

•First step: Isolate poly A+ mRNA with oligo (dT) cellulose

cDNA Library preparation

Cloning vectorsCloning vectors

• Cloning vectors are carrier DNA molecules. Four important features of all cloning vectors are that they:

• (i) can independently replicate themselves and the foreign DNA segments they carry;

• (ii) contain a number of unique restriction endonuclease cleavage sites that are present only once in the vector;

• (iii) carry a selectable marker (usually in the form of antibiotic resistance genes or genes for enzymes missing in the host cell) to distinguish host cells that carry vectors from host cells that do not contain a vector; and

• (iv) are relatively easy to recover from the host cell.

PlasmidsPlasmids• Naturally occurring extrachromosomal

DNA • Plasmids are circular dsDNA • Plasmids can be cleaved by restriction

enzymes, leaving sticky ends • Artificial plasmids can be constructed

by linking new DNA fragments to the sticky ends of plasmid

Vectors -- the DNA carriersMust have a origin of replicationAllow the vector as well as the foreign DNA to amplify in the host cell

1) Plasmids

2) Phages

Origin of replication

Antibiotic-resistant genes

Allow the host to grow on selective mediaCan selectively amplify this specific vector in the host cell

Multiple cloning sites

Allow insertion of foreign DNA

PlasmidsPlasmids

• Recombinant DNA vectors:– Amplification of DNA fragment can be achieved in the cell using cloning

vectors: plasmid or bacteriophages– Plasmid

Small circular DNA in bacteria or yeast cellsAccumulate 1-5 kb inserts

LacZ encodes -galactosidaseLacl – encodes factor controling transcription of lacZ

Bacteriophage Bacteriophage λλ (Lambda) (Lambda)•For cloning inserts of 10-20 Kb•Plasmid libraries hold up to 10 kb inserts

Bacteriophage Bacteriophage λλ (Lambda)Life (Lambda)Life CycleCycle

•Lytic Cycle:Production of progeny

•Lysogenic Cycle: Integration into bacterial chromosome

BACs: Bacterial Artificial BACs: Bacterial Artificial ChromosomesChromosomes

•Based on P1 bacteriophage, the F plasmid and the lacZ region of pUC plasmids

•It’s a low copy number plasmid•Carries 50-300kb fragments

BACs: Bacterial Artificial BACs: Bacterial Artificial ChromosomesChromosomes

YACs:Yeast Artificial YACs:Yeast Artificial ChromosomesChromosomes

• Transformation• Electroporation• Protoplast

fusion

DNA can be inserted into a cell DNA can be inserted into a cell by:by:

• Microinjection• Gene gun

DNA can be inserted into a cell DNA can be inserted into a cell by:by:

Bacterial transformationIntroduction of DNA into bacteriaSpontaneous uptake – low probabilityE. coli – cells treated with CaCl2Less than 1 of 103 cells acquire a plasmidSelection of transformed cells:

resistance to antibioticsusing chromogenic substances

Antibiotics: molecules produced by microorganism that kill other microorganismpeniciline, tetracycline, ciplroflaxine – inhibits gyrase in the complex with DNA – inhibits DNA replication

Chromogenic substances:

ScreeningScreening• The medium in this

petri dish contains the antibiotic Kanamycin

• The bacteria on the right contain Kanr, a plasmid that is resistant to Kanamycin, while the one on the left has no resistance

• Note the difference in growth

PropagationPropagation• Once colonies are

identified, they are cultured in broth to increase numbers and therefore the amount of DNA

• Samples are also prepared for storage at -80 degrees. They can be kept for many years this way.

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Agricultural ApplicationsAgricultural ApplicationsHerbicide

resistance -Broadleaf plants have been engineered to be resistant to the herbicide glyphosate

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Agricultural ApplicationsAgricultural ApplicationsPest resistance

-Insecticidal proteins have been transferred into crop plants to make them pest-resistant -Bt toxin from Bacillus thuringiensis

Golden rice-Rice that has been genetically modified to produce -carotene (provitamin A)

-Converted in the body to vitamin A

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Agricultural ApplicationsAgricultural Applications

Adoption of genetically modified (GM) crops has been resisted in some areas because of questions about:

-Crop safety for human consumption-Movement of genes into wild relatives-Loss of biodiversity

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Agricultural ApplicationsAgricultural ApplicationsBiopharming

-Transgenic plants are used to produce pharmaceuticals

-Human serum albumin-Recombinant subunit vaccines -Against Norwalk and rabies

viruses-Recombinant monoclonal antibodies -Against tooth decay-causing

bacteria

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Transgenic MammalsTransgenic Mammals

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• The insertion of genetic material into human cells for the treatment of a disorder

Medical Applications

Strategy for a subunit vaccine for herpes simplex

Recombinant DNA Recombinant DNA Vaccines?Vaccines?

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Gene TherapyGene Therapy

Treatment of SCID (severe combined immunodeficiency). SCID affects the maturation of immune cells that develop in bone marrow. SCID sufferers lack the enzyme ADA (adenosine deaminase).