Biotechnology & Recombinant DNA

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Biotechnology Recombinant DNA
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Recombinant DNA
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What is biotechnology?

• Using living microorganisms or cell components to
  make products
• Often via genetic engineering and using
  recombinant DNA

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What is recombinant DNA?

• Recall happens naturally
• Transposons
• Researchers can also produce it
• Use bacterial plasmids or viruses
  (bacteriophages)
• Uses
• Bacteria make insulin
• yeast help make components of Hepatitis B vaccine

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How is recombinant DNA made?

• Inserting foreign DNA into a bacterial cell
• Use restriction enzymes, plasmids, ligase and
  bacterial host
• Restriction enzymes
• Defense against viruses
• Clone gene or make gene product

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What are restriction enzymes?

• Naturally occur in bacteria
• Bacteria use these to combat viral infection
• Bacteria DNA uneffected because some is
  methylated
• Sequence-specific
• cloning animation

This can be used as a vector
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What is a vector?

• Different from a disease vector!
• plasmid animation
• Plasmid or virus
• Used to insert DNA into host cell
• Must be able to self-replicate!
• Must be small so not fragile
• Both types of vectors can allow researchers to
  clone DNA
• But theres another approach to DNA amplification

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PCR making copies of DNA
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What is PCR?

• Stands for _____________
• Need primers of about 20 bp to start
• DNA polymerase doesnt start reaction, only
  lengthens
• Primers recognize regions which flank target gene
• Ingredients
• primers
• DNA polymerase from TAC (Thermus aquaticus)
• Individual nucleotides
• Can you go over that one more time? PCR
  animation

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Why would you use PCR?

• Detect small amounts of DNA
• Can you think of examples?
• Get in groups and discuss!
• Forensics
• Infectious agents
• Gene mapping
• Human Genome Project
• Taxonomy and systematics studies
• Cancer and study of other human diseases
• Sequencing of rRNA and mRNA via cDNA

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Lets take a brief look at some of these
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DNA fingerprinting
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What is gel electrophoresis?

• Sorting DNA segments by size
• Process is called Southern Blotting

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What is Southern blotting?

• After gel electrophoresis
• Filter paper blots DNA off
• Radioactive probes added
• Autoradiography pinpoints sequence
• Southern Blot animation

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When would you use gel electrophoresis?

• DNA fingerprinting
• Restriction enzymes create restriction fragment
  length polymorphisms (RFLPs)

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What does DNA fingerprinting look like?
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Is this the only way to look at DNA?

• Nowe can sequence it, too!
• Sanger sequencing
• animation

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Cloning
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How do researchers get the DNA they want to clone?

• Gene libraries
• Either plasmid or phage libaries
• Fine for prokaryotic DNA
• Problem with eukaryotic DNA

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So whats the problem?

• Eukaryotic DNA has introns
• Must make complementary DNA (cDNA)
• Use reverse trasncriptase
• cDNA animation
• Now DNA can be inserted

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What do they do with the cloned DNA?

• Lots of different things!

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What are bioreactors?

• Using bacteria to produce gene products
• Insulin diabetes
• Human growth hormone
• Cellulase (break down cell wall for animal feed)
• Factor VIII hemophilia

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What are plant GMOs?

• Genetically modified organisms
• Transgenic plant or animal
• Bioreactors filled with these
• Plants
• Cotton, corn, potato to make pest resistant
• Soybeans resistant to common herbicide
• Some corn, cotton are herbicide and pest
  resistant
• Could produce human hormones, clotting factors,
  antibodies on seeds in future

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What are animal GMOs?

• Foreign genes into embryos
• produce animals that manufacture human hormones,
  etc. gene pharming
• Blood clotting factor goats
• Sheep milk with human alpha-1-antitrypsin (used
  to treat heritable emphysema)
• 1997, Dolly
• Since then, cloned sheep, cows, goats, mice
• Humans moratorium

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What is gene therapy?

• Insertion of genetic material into human cells to
  treat a disorder
• Use retrovirus to insert normal gene into cell
• Healthy genes to make up for faulty genes
• Severe combined immunodeficiency syndrome
• 1990, girl received normal gene in white blood
  cells
• Using genes to treat other illnesses