LEQ: How do we splice new genes into DNA?

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LEQ How do we splice new genes into DNA?

• 12.1 to 12.7 and 12.18

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Recombinant DNA technology

• This is a set of lab techniques for combining
  genes from different sources even different
  species into a single DNA molecule
• Began with the study of Eschericia coli (E. coli)

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Terms for recombinant DNA technology

• Gene Cloning the production of multiple copies
  of a gene
• Genetic Engineering the direct manipulation of
  genes for particle purposes
• Biotechnology the use of living organism (often
  microbes) to perform useful tasks

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Restriction Enzymes

• Bacterial enzymes that cut foreign DNA original
  purpose was a defense mechanism of bacterial to
  protect against foreign DNA (phage DNA)
• Restriction enzymes are now used to cut DNA
  molecules in reproducible ways
• These enzymes produce 2 different kinds of ends
• Blunt ends these enzymes cut straight through
  the double strand of DNA produces restriction
  fragments with no overlapping single strands
  fragments cannot bond with other pieces of DNA
• Sticky ends these enzymes have a staggered cut
  resulting in fragments with single stranded ends
  which can bond with complementary DNA

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STICKY OR BLUNT?

• Sticky

• Blunt

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Plasmids

• small circular ring of DNA found in prokaryotes
  and yeast
• Important sites of plasmid
• 1. origin of replication
• 2. genetic marker (i.e.
• antibiotic resistance)
• 3. restriction enzyme
• cut sites

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Restriction Enzymes Recombinant DNA

• Restriction enzymes have specific recognition
  sites
• Scientists use a specific enzyme to cut out a
  specific gene and a specific plasmid
• The sticky ends of the gene and the plasmid will
  compliment each other allowing scientists to join
  the two
• Use DNA Ligase to join two DNA types to produce
  recombinant DNA

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Cloning Recombinant DNA

1. Identify a restriction enzyme that will cut out
   gene of interest and cut open the plasmid
2. Isolate DNA from 2 sources (making sure the at
   the plasmid has a genetic marker)
3. Cut both types of DNA with the same restriction
   enzyme
4. Mix the 2 types of DNA to join them through
   complementary base pairing Add
5. DNA ligase to bond DNA covalently producing
   Recombinant DNA
6. Incubate bacteria at 42 C with calcium chloride
   bacteria become competent / permeable - so that
   the bacteria will take in the plasmid
   (TRANSFORMATION)
7. Use a genetic marker to identify bacteria with
   the recombinant plasmid
8. Clone bacteria

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Clone Storage via Genomic Library

• Plasmid Libraries and Phage Libraries are created
  by cutting a genome into fragments
• Genome fragments are used to create recombinant
  DNA
• Recombinant DNA is then either stored in a
  bacterial culture or a phage culture

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Reverse Transcriptase used to make genes for
cloning

1. Transcribe DNA into RNA in the nucleus
2. RNA splicing occurs removing introns
3. Isolate mRNA from the cell and add reverse
   transcriptase to synthesize a new strand of DNA
4. The mRNA is digested
5. Synthesize 2nd complimentary strand using DNA
   polymerase creating cDNA

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Whats the difference?

• DNA

• cDNA

• Contains introns - must be edited to be get to
  the DNA that codes for a gene

• No introns the actual DNA that codes for a
  particular gene

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Genetically Modified Organisms

• Genetically modified organism an organism that
  acquires one or more genes by artificial means
  (gene may or may not be from a different species)
• Transgenic organism organism that contains a
  gene from another species

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Recombinant DNA Applications

• Bacteria are protein factories. E. coli is the
  primary bacteria used cheap and easy to maintain
  cultures and produce proteins (pharmaceutical
  factories)
• Products
• Chymosin (used for cheese production), Human
  Insulin Human Growth Hormone Factor VIII
  Hepatitis B vaccine Diagnosis of HIV

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Recombinant DNA Applications

• The yeast Saccharomyces cerevisiae is commonly
  used when eukaryotic cells are needed.
• Yeast serve as a good vector for human genomic
  libraries

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Recombinant DNA Applications

• Plants
• By genetically modifying the genome of a plant
  scientists have been able to increase the
  nutritional value of crops (i.e. Golden Rice
  contains daffodil gene allowing it to produce
  beta-carotene) scientists have been able to
  genetically modify plants that are drought
  resistant, pesticide resistant, larger in size,
  and that have a longer shelf life.

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Recombinant DNA Applications

• Mammals
• Recombinant DNA technology is used to add a human
  gene for a desired human trait (protein) to the
  genome of a mammal in such a way that the genes
  products, such as antithrombin (protein that
  prevents blood clots), are secreted in the milk
  of the animal Transgenic mammals allow
  scientists to model human diseases and find
  treatments to the diseases Transgenic pigs may
  serve as human blood and organ donors Transgenic
  cattle fish have been engineered to be larger
  in size providing more meat

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DNA Technology pharmaceuticals and Medicine

• Therapeutic Hormones human insulin produced by
  bacteria (no longer use insulin from cadavers or
  pigs)
• Diagnosis Treatment of disease identify
  disease causing alleles tailored treatments
• Vaccines create harmless variants of pathogen
  to stimulate the immune system