Genetic Engineering Recombinant DNA (rDNA) Technology rDNA

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Genetic 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)

• 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 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 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 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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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)

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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)

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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

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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

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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

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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

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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)
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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 as well
• 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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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 but interrupts ß-galactosidase)
• Blue-white screening

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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)

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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 cant grow on
  2nd plate

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Replica plating diagram
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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
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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

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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

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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

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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