Download - Genetic engineering
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Genetic EngineeringRecombinant DNA (rDNA) TechnologyGenetic EngineeringRecombinant DNA (rDNA) Technology
• rDNA technology involves cloning DNA by cutting & pasting DNA from different sources
• Restriction enzymes & DNA ligases are important enzymes for this process
• DNA ligases join together adjacent DNA fragments
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Genetically Modified Organisms (GMOs)
Genetically Modified Organisms (GMOs)
• GMOs are organisms that have had genetic material removed and/or inserted in order to change a particular trait or traits of the organism.
• The process is called gene splicing or genetic engineering
• Organisms produced by transplanting genetic materials between different types of organisms are called transgenic organisms.
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Transgenic Organism ExamplesTransgenic Organism Examples
• Genes from bacteria are spliced into corn and cotton to make them less susceptible to insect damage
• Human growth hormone implanted into mice & other animals so that it can be harvested
• ANDi (first transgenic monkey) is a rhesus monkey carrying GFP protein, showing foreign gene can be inserted into primate chromosome
• May lead to primate models of human diseases
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Restriction enzymesRestriction enzymes• Restriction enzymes are DNA-cutting enzymes that are found in bacteria
• They are also called endonucleases (cut within DNA sequences)
• Microbiologists from 1960s discovered that some bacteria are protected from destruction by viruses because they cut viral DNA, restricting viral replication
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Restriction enzymes Q & ARestriction enzymes Q & A
In 1970, Hamilton Smith isolated HindIII (1st restriction enzyme well characterized and used for DNA cloning), which comes from Haemophilus influenzae.
They are named based on genus & species of bacteria it was isolated from. (EcoRI = Escherichia coli, RY13).
They cut DNA by cleaving phosphodiester bonds (in sugar-phosphate backbone) that join adjacent nucleotides
Which was the first one well understood?
How are they named?
How do they work?
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SpecificitySpecificity
• Restriction enzymes show specificity for certain substrates (DNA in this case)
• They recognize, bind to, and cut DNA at specific sites called restriction sites (recognition site)
• Usually a 4-base pair or 6-base pair cutter
• Restriction sites are palindromes (reads same forward & backwards on opposite strands)
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Restriction cutsRestriction cuts
• Some cut DNA to create fragments with overhanging single-stranded ends (sticky ends or cohesive ends), while others create fragments with non-overhanging ends (blunt ends)• Enzymes that create sticky ends are favored for cloning experiments since the DNA fragments can be easily joined together
• DNA from any source can be digested (as long as it has the specific restriction site)
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GE ApplicationGE Application
• In 1972, Paul Berg joined DNA from E.coli and a primate virus called SV40
• He cut both with EcoRI (restriction enzyme)
• He then added fragments to tube with DNA ligase
• This became 1st recombinant DNA molecule
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PlasmidsPlasmids
• Plasmid DNA is circular form of self-replicating DNA that scientists can manipulate to carry and clone other pieces of DNA
• Found primarily in bacteria
• Considered extrachromosomal DNA because they are present in addition to chromosomes
• They are small (~1000 - 1400 base pairs) in size
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VectorsVectors
• Plasmids can be used as vectors (pieces of DNA that can accept, carry, and replicate other pieces of DNA)
• 1st plasmid vector pSC101
(SC = Stanley Cohen, pictured left)
• Contained gene for tetracycline (antibiotic) resistance and restriction sites for several enzymes
• rDNA animation
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VectorsVectors
• Cohen & Boyer (pictured left) awarded patents (1980) for pSC101 and gene splicing & cloning technologies
• Major concern at the time was the thought of recombinant bacteria leaving the lab
• Boyer joined forces with Robert Swanson (venture capitalist) to create Genentech in an effort to commercialize these technologies
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Vector FeaturesVector Features
Modern plasmid DNA cloning vectors usually consider 6 desirable features:
1. Size (must be small enough to separate easily)
2. Origin of replication (ori) - DNA sequence at which replication is initiated
3. Multiple cloning site (MCS) - a stretch of DNA with recognition sequences for common restriction enzymes (Engineered into plasmid so that digestion does not result in loss of DNA fragment)
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Vector FeaturesVector Features
4. Selectable marker genes - allow for selection and identification of transformed bacteria• Most common selectable markers are antibiotic resistance.
• Lac z gene widely used (gene of interest inserted within lac z gene)
• Plated on X-gal (substrate similar to lactose but turns blue when cleaved by ß-gal); so, recombinant bacteria turn blue & nonrecombinant are white
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SelectionSelection
• Selection is a screening process designed to facilitate the identification of recombinant bacteria while preventing growth of nontransformed bacteria (or those containing plasmid without foreign DNA)
• Blue-white screening is becoming more popular (uses ß-galactosidase)
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Antibiotic selectionAntibiotic selection
• Antibiotic selection uses a plasmid vector with genes encoding resistance to 2 different antibiotics, usually ampicillin (ampR) and tetracycline (tetR)
• Foreign DNA inserted into one of the 2 antibiotic resistance genes (disrupts gene - preventing protein)
• Transformed cells are plated to an agar plate with no antibiotic or plate with one (ampicillin)
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Replica platingReplica plating• Replica plating uses sterile pads pressed against colonies on plate (cells adhere to make an exact copy)
• Then pad is placed on 2nd replica plate containing 2nd antibiotic (tetracycline)
• Nontransformed bacteria cannot grow in presence of either antibiotic without plasmid
• Compare plates since recombinant can’t grow on 2nd plate
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Replica plating diagramReplica plating diagram
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Vector FeaturesVector Features
5. RNA polymerase promoter sequences - place where RNA polymerase binds to begin transcription
6. DNA sequencing primer sequences - known sequence that allows sequencing of cloned DNA fragments that have been inserted into the plasmid
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Types of VectorsTypes of Vectors
One primary limitation of bacterial plasmids as vectors is the size of DNA fragments (usually cannot exceed 6-7kb: 6000-7000 base pairs).
• Bacteriophage vectors
• Expression vectors
• Bacterial artificial chromosomes (BACs)
• Yeast artificial chromosomes (YACs)
• Tumor-inducing (Ti) vectors
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Gene TransferGene Transfer• Cohen discovered that plasmid DNA enters a bacterial cell (transformation) treated with calcium chloride, chilled on ice, then briefly heated
• A more recent transformation technique is electroporation (brief pulse of high-voltage electricity to create tiny holes in bacterial cell wall allowing DNA to enter)
• Cells that have been treated for transformation (so they are more receptive to take up DNA) are called competent cells
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BiolisticsBiolistics• Sometimes, biolistics are used in order to have foreign DNA enter a cell
• DNA is blasted into the cell using tiny bullets composed of tungsten or gold particles with DNA attached
• Done with a gene gun (aka bioblaster)
• Can be used on bacteria, yeasts, & mammalian cell lines
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National Institutes of Health (NIH)
National Institutes of Health (NIH)
• Concerns arose because of new techniques
• In 1975, NIH formed the Recombinant DNA Advisory Committee (RAC) to evaluate risks and establish guidelines for rDNA technology