GENETIC MODIFICATIONS

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GENETIC MODIFICATIONS

• Genetic engineering altering the sequence of DNA
• Ideas established in early 70's by 2 American
  researchers, Stanley Cohen (worked with plasmids)
  and Herbert Boyer (restriction endonucleases)
• Initially had no commercial applications for
  their experiments, but things changed quickly.
• In 1976 Boyer cofounded Genetech, first biotech
  company to go public on the stock market.

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• 1978 somatostatin became the first human hormone
  produced by this technology
• Other examples
• Insulin over 90 diabetics are reliant on human
  insulin supplied by bacteria.
• Somatropin used to treat human growth
  deficiency, from dwarfism, Turner's syndrome,
  also used for AIDS-associated wasting syndrome now

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BIOTECHNOLOGY

• Biotechnology involves the manipulation of DNA
  and protein synthesis.
• Molecular biologists analyze and alter genes and
  their respective proteins

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Examples

• Genetic screening scanning for genetic mutations
• Gene therapy the alteration of a genetic
  sequence in an organism to prevent or treat a
  genetic disorder by creating working proteins.
• Transgenic plants inserting genes to provide new
  proteins, giving plants new properties
• DNA fingerprinting analyzing pattern of bands
  that are unique to an individual.
• Human Genome Project...

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Biotech Tools

• The tools the scientists use are very specific to
  DNA and its environment.
• The DNA first has to be cut out of the source
  organism
• The DNA has to be isolated
• DNA can then be introduced into host DNA

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Recombinant DNA

• Recombinant DNA is DNA from one source organism
  being put into the DNA of a host organism.

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1) Cutting Out DNA

• Restriction Endonucleases / Enzymes are naturally
  occurring enzymes that act like a pair of
  molecular scissors to cut DNA in a predictable
  and precise manner, at a specific nucleotide
  sequence called a recognition site.

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Discovery

• Hamilton Smith, John Hopkins University, won the
  Nobel Prize in 1978 for discovering restriction
  enzymes in bacteria.
• He found their main purpose was to cut foreign
  DNA that tried to invade a bacterial cell (ie DNA
  from a virus).

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Naming System

• Restriction enzymes are named according to the
  bacteria from which they originate.
• BamHI is from Bacillus amyloliquefaciens, strain
  H. The I indicates it was the first endonuclease
  isolated from that strain.

EcoRI -  from Escherichia coli BamHI - from
Bacillus amyloliquefaciens HindIII - from
Haemophilus influenzae (the one H. Smith
found)PstI -  from Providencia stuartii Sau3AI
- from Staphylococcus aureus AvaI -  from
Anabaena variabilis
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Recognition sites

• 4 8 base pairs in length.
• Palindromic both strands have the same sequence
  when read in the 5' to 3' direction.
• Ex. HincII recognizes the following sequences
• 5'-G T C GA C-3'      5'-G T T G A C-3'      5'-G
  T C A A C-3'      5'-G T T A A C-3'
• 3'-C A G C T G-5'   3'-C A A C T G-5'      3'-C
  A G T T G-5'     3'-C A A T T G-5'

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• The restriction enzyme EcoRI binds to
  5'-GAATTC-3' 3'-CTTAAG-5'
• EcoRI breaks the phosphodiester bond between G
  and A,
• then it pulls apart the two strands by breaking
  the H-bonds between the complementary base pairs.
• Produces what are called sticky ends (unpaired
  nucleotides at each end).

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Sticky vs. Blunt

• Other restriction enzymes like AluI produce blunt
  ends, or ends with no overhang.
• Sticky ends are usually more helpful to molecular
  biologists as they can easily be joined with
  other DNA fragments cut by the same restriction
  enzyme.
• Blunt ends are harder to fuse to a foreign DNA
  molecule.

• p281 1-5

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• A host must protect its own DNA from
  endonucleases.
• Methylases are enzymes that place a methyl group
  (CH3) on recognition sites
• This prevents the restriction enzyme from
  cleaving the DNA at that spot.
• Host DNA is methylated, but foreign DNA is not,
  so it can be cut by the host cell's restriction
  enzymes.

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2)Isolating DNA Fragments

• Scientists make use of restriction endonucleases
  to cleave DNA into smaller fragments
• Gel electrophoresis is used to isolate the
  required gene segment from the rest of the DNA

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Gel Electrophoresis

• The fragments of DNA will be run through a porous
  agarose gel using electricity.
• The fragments of DNA are pulled through pores in
  the gel due to their negative charge.
• Smaller fragments will move faster than larger
  because they can fit through the pores better.

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http//www.stanford.edu/group/hopes/diagnsis/gente
st/f_s02gelelect.gif
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Steps

• Solutions of fragments are placed in wells
  (depressions at one end of the gel)
• The DNA is mixed with a dye so it will be seen as
  it moves through the gel.
• Markers are usually put in the first well, These
  are pieces of DNA whose size is known. They help
  determine the length of the unknown DNA
  fragments.

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• The gel is submerged in a buffer solution and
  connected to a power source.
• The anode will be at the top and the cathode at
  the bottom. DNA is negatively charged, it will
  move away from the anode to the cathode.
• The power source is only left on for a set amount
  of time, so the fragments dont move all to the
  end or run off the gel, you want them separated
  on the gel.

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VIEWING THE GEL
http//www.mcps.k12.md.us/departments/intern/stp/i
mages/gel_electrophorsis.jpg
http//www.life.uiuc.edu/molbio/geldigest/fullsize
/geldraw.jpg

• The gel is stained with ethidium bromide which
  will cause the gel to fluoresce under UV light.
• The band of the DNA fragments can be seen and the
  researcher is able to compare samples from
  various sources or isolate a DNA fragment they
  want to purify.

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3) INTRODUCING FOREIGN DNA INTO A HOST PLASMIDS
and TRANSFORMATION

• Plasmids are
• small (1000 to 200 000bp in length),
• circular DNA molecule
• independent of the bacterial chromosome.
• Plasmid DNA can be replicated using the bacterial
  cells machinery.

http//www.rpgroup.caltech.edu/courses/PBL/images_
dnascience/pZ20Plasmid.gif
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Plasmids

• Beneficial because they often contain important
  genes such as antibiotic resistance, heavy metal
  protection.
• Plasmids are used by biologists to incorporate
  genes they want replicated or transcribed/translat
  ed in vast amounts in little time into bacterial
  cells.
• Vector vehicle used to introduce DNA into a host
  cell, ie a plasmid or virus.

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3) INTRODUCING FOREIGN DNA INTO A HOST

• If we can cut genes out, we must be able to join
  them to foreign DNA.
• When sticky ends join together, DNA ligase
  recreates the phosphodiester bonds.
• Blunt ends cannot be joined by our own DNA
  ligase, they must be joined by ,
  an enzyme from the T4 bacteriophage (virus).

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STEPS

• Restriction enzymes are used to cut out the gene
  from the original cell AND to open the bacterial
  plasmid.
• Once the foreign gene is isolated it can then be
  inserted into the plasmid. The plasmid is now
  considered recombinant DNA.

http//employees.csbsju.edu/hjakubowski/classes/ch
331/dna/plasmid.gif
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TRANSFORMATION

1. The recombinant DNA is then introduced into a
   bacterial cell. Sometimes a host cell must be
   manipulated to take up the foreign DNA plasmid.

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• Transformation introduction of foreign DNA
  (usually by plasmid or virus) into a bacterial
  cell.
• Host cell cell that has taken up foreign plasmid
  or virus and whose cellular machinery is being
  used to express the foreign DNA.
• Competent cell cell that readily takes up
  foreign DNA.

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4) Selection and Cloning

• Cells that have been successfully transformed
  must be isolated (usually by antibiotic
  resistance)
• The vectors used for cloning usually carry an
  antibiotic-resistance gene. Growth of colonies
  on media containing the antibiotic indicates
  successful transformation.

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Cloning

• Colonies are isolated from media and grown in
  culture to produce multiple copies (clones) of
  the recombinant DNA
• When the bacteria replicates the recombinant DNA
  plasmid, the new gene product will be formed
  multiple times (ie. the gene is cloned).

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PCR another means of copying DNA in large
numbers

• stands for Polymerase Chain Reaction,
• developed in the late 1980's by Kary Mullis
  awarded Nobel Prize in Chemistry in 1992.
• Does not require a plasmid. The fragment is
  copied directly.
• Useful for forensic criminal investigations,
  medical diagnosis, genetic research. Only small
  amounts of DNA are needed.

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PCR Process

• PCR is amplification of a DNA sequence by
  repeated cycles of strand separation and
  replication in the laboratory (DNA photocopying).
• After about 30 cycles more than 1 billion copies
  of the targeted area will exist (230).

http//users.ugent.be/avierstr/principles/pcrcopi
es.gif
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Steps of PCR

• Strands are separated using heat
• DNA primers, synthesized in the lab, are created
  to complement the start of the target area to be
  copied.
• Temp is decreased and the primers anneal
• Taq polymerase (from bacteria) creates new
  strands of target area
• Sequence is repeated over and over on each of the
  new strands built

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Restriction Fragment Length Polymorphism (RFLP)

• Entire genome is subjected to restriction enzyme
  digestion
• DNA run on an agarose gel, using gel
  electrophoresis
• Single stranded DNA transferred to a membrane

http//homepage.smc.edu/HGP/images/rflp.gif
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RFLP

• ssDNA hybridized with radioactive probes for
  specific regions (such as alleles or areas known
  as variable number tandem repeats, that lead to a
  specific disease).
• An X-ray film is developed, called an
  autoradiogram, and the pattern can then be used
  to identify a suspect, or detect a genetic
  mutation.

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SEQUENCING DNA

• Sanger dideoxy method uses DNA replication and
  dideoxy nucleotides to determine the
  complementary strand.
• Developed by Frederick Sanger and colleagues at
  Cambridge University in Great Britain in 1977.
  They used it to sequence the genome of a
  bacteriophage (viral DNA) 5386 base pairs long.

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Sanger dideoxy method

• Dideoxy nucleotides are missing the -OH group on
  carbon 3 and therefore inhibit the process of
  replication.
• Every time one is added, the process stops and
  only small sequences are created.

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• These sequences can be run on a gel, and since
  they will run from shortest to longest, you can
  actually read the sequence by knowing which
  dideoxy nucleoside was used and therefore stopped
  replication at each point.

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Fluorescent Detection of Oligonucleotides

• The Human Genome Project used a similar method,
  but also included fluorescence on each dideoxy
  nucleoside, so the A, G, T and C's lit up as
  different colours.

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• A computer reads the sequence from gel
  electrophoresis.
• Thousands of sequencers worked 24 hours a day, 7
  days a week to decipher 3 billion base pairs.