Chapter 23 (Part 1)

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Chapter 23 (Part 1)

• Recombinant DNA Technology

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Recombinant DNA Technology

• Methods for isolating, manipulating, and
  amplifying identifiable DNA sequences.
• Allows us to study the structure and function of
  individual genes.
• Allows for the directed genetic manipulation of
  organism (modify gene function, insert novel
  genes)

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Cloning

• Clone a collection of molecules or cells, all
  identical to an original molecule or cell
• To "clone a gene" is to make many copies of it -
  for example, in a population of bacteria
• Gene can be an exact copy of a natural gene
• Gene can be an altered version of a natural gene
• Recombinant DNA technology makes it possible
• Allows for in vitro manipulation of a individual
  gene

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Tools Needed for Cloning(Think of it as a
cutting and pasting process)

• cDNA or genomic library (source of DNA to cut)
• Plasmid (where you want to paste it)
• Restriction enzymes (scissors)
• DNA ligase (paste)
• E. coli (biological machine needed to amplify
  DNA)

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Plasmids

• Naturally occurring extrachromosomal DNA
• Self replicating circular double stranded DNA
  molecules that have their own origin of
  replication
• Usually present in multiple copies per cell
• Plasmids can be cleaved by restriction enzymes,
  leaving sticky ends
• Artificial plasmids can be constructed by linking
  new DNA fragments to the sticky ends of plasmid

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Cloning Vector

• Required features
• Origin of replication
• Selectable marker
• Screenable marker for recombinant molecules
• Cloning sites

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Restriction Enzymes

• Bacteria protect themselves from attack by
  viruses and other bacteria using a
  restriction/modification system.
• Allows bacteria to recognize and destroy foreign
  DNA
• Bacteria contain DNA methylases that modify their
  chromosomal DNA at specific sequences.
• Also contain restriction endonucleases that
  recognize and cleave these same sequences when
  they are not methylated

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Restriction Modification System
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Restriction Enzymes

• Type I Contain methylase and endonulcease
  fuctions. Require ATP for hydrolysis and
  S-adenosylmethionine for methylation
• Type II contain only endonulcease function,.
  Does not require ATP for hydrolysis.
• Both types recognize palindrome sequences
  (sequences that read the same if read forward or
  backwards e.g. BOB or DEED

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Type II Restriction Enzymes

• Names use 3-letter italicized code
• 1st letter - genus 2nd,3rd - species
• Following letter denotes strain
• EcoRI is the first restriction enzyme found in
  the R strain of E. coli

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Sticky-end cutter
Blunt-end cutter
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Restriction Enzymes

• Restriction enzymes can recognize specific 4
  base, 6 base, 8 base sequences.
• The probability that a given piece of DNA will
  contain a specific restriction site is n4
• n the number of bases in the restriction site
• So for a 6 base cutter (64), you would expect to
  find your site every 1300 base pairs. So in a
  10,000 bp fragment there is likely to by 7 or 8
  restriction sites corresponding to your enzyme.
• You can characterize DNA fragments using gel
  electrophoresis

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T4 DNA Ligase
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Transformation

• All of the previous steps were performed in
  vitro.
• We have generated a very small amount of a
  recombinant plasmid
• Need to amplify in bacteria to get enough to work
  with.
• Transformation process to mobilize DNA into
  bacterial host
• Select for transformed bacteria on specific
  antibiotic that corresponds to the antibiotic
  resistance gene present on the plasmid

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How to produce a recombinant protein
0.1 to 1 of cellular protein
10 to 70 of cellular protein
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Cloning a gene from a DNA libraries

• Any particular gene may represent a tiny, tiny
  fraction of the DNA in a given cell
• Can't isolate it directly
• Trick is to find the fragment or fragments in the
  library that contains the desired gene

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cDNA
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cDNA Library
cDNA
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Library Screening

• DNA probe hydridization
• Requires that you know the protein or amino acid
  sequence of the gene of interest.
• Need to denature (make single stranded) and
  immobilize the DNA from each clone of the library
  to a filter (nitrocellulose or nylon)
• Make a labeled single stranded DNA/RNA probe (can
  use radioactive of fluorescent analogous of
  specific nucleotide triphosphates)
• Labeled single stranded DNA/RNA fragments will
  base pair (hydridize) with the target DNA on the
  filter
• Identify clones that are labeled.

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DNA hydridization screening for specific gene

• Requires that you know something about the gene
  sequence
• Can get sequence information form purified protein

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Now that we have the gene, what do we do with it?

• We could use it make a lot of protein in a
  microbial protein expression system
• We could use it to genetically manipulate
  organisms
• We could use it as a diagnostic tool

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Why use recombinant Proteins?

• Proteins are often only available in small
  amounts in a given tissue
• Tissue sources may not be readily available
• It is time consuming and expensive to purify
  protein from tissues
• It is difficult to obtain absolutely pure protein
  

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Insulin

• Was first purified from human pancreas from
  cadavers and then from pig pancreas.
• Genentec expressed insulin gene in microbial host
• Can grow microbes in large fermenters to produce
  unlimited supply of insulin.

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Recombinant proteins are also important to
research

• For enzyme analysis need pure protein
• For structural analysis need lots (milligram
  amounts) of very pure protein
• Need pure proteins to make diagnostic tools such
  as antibodies

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Genetic Modification of Higher Organisms

• Can introduce gene into animals and plants
• These modified organism are powerful research
  tools to study the effect of a specific gene
  product on metabolism, development etc.
• Has also been used to develop improved
  agricultural products

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Genetically Engineered Salmon Is Bigger Better?
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http//www.agwest.sk.ca/sabic_index_tp.shtml
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Plant Genetic Engineering

• Improved Agricultural Production
• Herbicide Resistance
• Pest Resistance
• Improved Nutrition
• Vitamins - Golden Rice, Vitamin E
• Increase essential Amino Acid Content
• Chemical Synthesis
• Bio-plastics
• Bio-diesel
• Lubricants/detergents
• Rubber

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GMO Concerns

• Ecological Concern
• Potential Food Allergens
• Antibiotic Resistance

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GMO Benefits

• Lower application of herbicides and pesticides
• Creation of foods with increased nutrition
• Creation of bio-based alternative to petroleum
  based products

http//www.colostate.edu/programs/lifesciences/Tra
nsgenicCrops/