Genetic engineering
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Genetic engineering

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Genetic engineering Genetic engineering Presentation Transcript

  • Genetic Engineering Recombinant 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
  • 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.
  • Transgenic 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
  • Restriction 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
  • Restriction 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?
  • Specificity
    • 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)
  • Restriction 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)
  • GE 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
  • Plasmids
    • 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
  • Vectors
    • 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
  • Vectors
    • 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
  • Vector 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)
  • Vector 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
  • Selection
    • 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)
  • Antibiotic 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)
  • Replica 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
  • Replica plating diagram
  • Vector 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
  • Types 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
  • Gene 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
  • Biolistics
    • 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
  • 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