Genetic Engineering

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Genetic Engineering

• An Overview

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What is it???

• Applied techniques of genetics and biotechnology
  (Wet lab procedure). Much trial and error.
• Involves the isolation, manipulation and
  reintroduction of DNA into cells or model
  organisms, usually to express a protein.
• DNA taken from one organism and inserted
  (transformed) into another (transgenic) organism
• Heritable, directed alteration of an organism.
  Altering DNA or adding new DNA allows us to
  change the characteristics of a cell or cells.

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Why do it?

• It aims to introduce new characteristics or
  attributes physiologically or physically, such as
  making a crop resistant to a herbicide,
  introducing a novel trait, or producing a new
  protein or enzyme.
• For example

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How does it work?

• Detect gene within organism of interest
• Isolate and Splice out DNA
• Ligate into Vector of choice
• Introduce into bacteria
• Isolate colonies containing vector with gene of
  interest
• Grow up bacteria colony to produce multiple
  copies of gene which is expressed

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Isolating and splicing DNA of Interest

• Conventionally entire genome fragmented using
  Restiction Enzymes
• E.g. BamH1
• Hindiii
• Target Palindromic sequences
• AGGTACCT
• TCCATGGA

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• Can result in Staggered/Sticky ends
• AG GTACCT
• TCCATG GA
• Can result in Blunt ends
• AGGT ACCT
• TCCA TGGA
• Enzymes can create sticky ends from blunt ones

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• PCR can be used to specifically target gene of
  interest
• Enzymatic amplification of specific DNA fragment
  using repeated cycles of DNA denaturation, primer
  annealing and Chain extension
• Knowledge of full sequence not required
• Can produce large amount of copies from gtminute
  quantity of target DNA
• gtPartially damaged DNA

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Inserting Gene fragments into Vector of choice

• Many types of Vectors depending on needs
• Bacterial Artificial Chromosomes
• Yeast Artificial Chromosomes
• Plasmids
• Use RE to create complimentary sticky ends in the
  vector

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• Possible outcomes post digestion with R.E.
• Plasmid reforms
• Fragmented genomic DNA joins up with itself
• Plasmid and fragmented DNA for a hybrid
• Contains gene of interest
• Contains other gene (unfortunately, most times!!!)

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Insert into Bacteria/Host

• Allows expression of protein product
• Allows multiple copies of the gene to be made
• Several methods
• Heat shock
• Electric Pulse (electroporation)
• Microinjection/microprojectiles
• Viruses
• Lysosomes

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Identifying the right colony

• 3 levels of identification
• Bacteria which have taken up a plasmid
• Bacteria which have taken up a recombinant
  plasmid
• Bacteria containing the wanted gene
• Achieved by using antibiotic resistence genes and
  gene probes.

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Finally

• Once the correct colony is identified it is
  cultured and gene expression encouraged.
• Protein product can be harvested!
• However it is not always successful Much
  patience is required!!!

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Some examples of current applications of genetic
engineering

• Synthesis of insulin. Not always effective due to
  a eukaryotic protein being expressed in a
  prokaryotic cell.
• Introduction of resistance genes in crop plants
• Flavr Savr Tomato
• Gene therapy (e.g. Cystic Fibrosis)