Gene Cloning and PCR: Vectors

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Gene Cloning and PCR Vectors

• Dr. Jason Linville
• University of Alabama at Birmingham
• jglinvil_at_uab.edu

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History

• Before 1950, experiments suggested DNA was
  genetic material.
• Early 50s, DNA identified and structure
  discovered.
• 50s early 60s, genetic code cracked,
  transcription and translation described.

However, laboratory techniques to study these
newfangled genes stunk.
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History

• Early 70s, new methodology of recombinant DNA
  technology or genetic engineering developed.
• Involved use of gene cloning

Later

• 1985, Kary Mullis thinks up the polymerase chain
  reaction (PCR)

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Gene Cloning

• Fragment of DNA inserted into a vector.
• Result is a chimera or recombinant DNA molecule.

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Gene Cloning

• Vector carries gene into a host cell (usually
  bacteria)

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Gene Cloning

• Chimera and host cell multiplies, creating many
  copies of the gene.

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Gene Cloning

• Cell divisions create colony (clones) of
  identical host cells.
• Each cell contains one or more copies of
  recombinant DNA molecule

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Gene Cloning

• Once gene is cloned, information can be gathered
  about its structure and expression.

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Gene Cloning

• If multiple DNA fragments are available, how is
  only one isolated?

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Gene Cloning

• Usually, only one vector will enter a bacteria.

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Gene Cloning

• Q How is the desired colony identified?

A Different ways we will explore later.
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Polymerase Chain Reaction

• Like gene cloning, PCR results in the
  amplification of a region of DNA.

PCR is not done in living cells
PCR is done in test tubes with isolated DNA
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Polymerase Chain Reaction
PCR Hood
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Polymerase Chain Reaction
Thermal Cycler - amplifies DNA
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Polymerase Chain Reaction

• Mixture of DNA and reagents (DNA polymerase
  primers) heated to 95ºC

Heat breaks H-bonds DNA denatures
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Polymerase Chain Reaction

• Mixture is cooled (50-60ºC), allowing primers to
  reanneal to DNA based on primer sequence

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Polymerase Chain Reaction

• Temperature increased (72-75ºC), allowing DNA
  polymerase to attach and begin replication.

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Polymerase Chain Reaction
Cycle repeated 25-30 times

• Results in double stranded DNA fragments with
  primer sequences at their ends.

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Gene Cloning vs. PCR
Limitations of PCR

• In order to amplify an area, the sequences of the
  primer annealing sites must be known.
• Limited by length regions gt5 kb are difficult to
  amplify.

Gene Cloning is only way of isolating long genes
or unstudied genes.
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Gene Cloning vs. PCR
PCR only amplifies one area
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Gene Cloning
Vehicles for Gene Cloning

• In order to clone a gene, it must be transported
  into living cell.

Two mechanisms for transport

• Plasmid circular molecule of DNA

• Bacteriophages protein capsule injects DNA

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Gene Cloning gt Plasmids
Plasmids

• Independent circular DNA in bacteria

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Gene Cloning gt Plasmids

• Have origin of replication and can use host
  cells enzymes to replicate independently.

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Gene Cloning gt Plasmids

• Or possible to integrate into bacterial
  chromosome.

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Gene Cloning gt Plasmids
Plasmid Size

• lt10 kb useful for cloning natural plasmids can
  range up to 250 kb

Copy Number

• Number of plasmids per cell
• Range from 1 - gt50
• Ideally, higher copy number better

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Gene Cloning gt Plasmids

• Plasmids may contain antibiotic resistance genes.
• Allows for the selection of bacteria that contain
  the plasmid.

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Gene Cloning gt Plasmids

• When grown in antibiotic medium only bacteria
  with resistant plasmid will grow.

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Gene Cloning gt Plasmids
Plasmid Classifications

• Fertility Plasmids promote conjugal transfer of
  plasmids
• Resistance plasmids contain antibacterial
  resistance genes
• Col plasmids code for colicins, proteins that
  kill other bacteria

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Gene Cloning gt Plasmids

• Conjugation allows for transfer of plasmid copy.

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Gene Cloning gt Plasmids
Plasmid Classifications

• Degradive plasmids allow host to metabolize
  unusual molecules
• Virulence plasmids confer pathogenicity on host
  bacteria

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Gene Cloning gt Bacteriophages
Bacteriophages
M13
?

• Viruses that specifically infect bacteria.
• DNA (or RNA) surounded by protein coat
• Genes code for capsid and replication proteins

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Gene Cloning gt Bacteriophages gt ?
Bacteriophages (Infection)

• Lytic Cycle rapid infection resulting in lysis
  of cell and release of multiple bacteriophages.

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Gene Cloning gt Bacteriophages gt ?

• Phage attaches to outside of bacterium injects
  DNA into cell.

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Gene Cloning gt Bacteriophages gt ?

• Phage genome replicated with enzymes coded for
  by phage genome

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Gene Cloning gt Bacteriophages gt ?

• Capsid proteins synthesized phage particles
  assembled and released.

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Gene Cloning gt Bacteriophages gt ?

• Lysogenic phages For bacteriophage ?, DNA
  integrates in to bacterium genome.

• When released, enters lytic cycle.

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• ? has
• sticky ends

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Gene Cloning gt Bacteriophages gt M13

• Lysogenic phages For bacteriophage M13, phage
  particles assembled and released without lysing
  cell.

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Gene Cloning gt Bacteriophages gt M13
M13 Advantage

• Genes can be obtained in single strand form. This
  is very useful when sequencing gene.

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