Biotechnology

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Biotechnology

• Techniques and Applications

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GENES ARE THE RECIPES FOR PRTEINS
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Each codon places a different amino acid in the
protein.
The genetic code
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Decoding DNA to make proteins
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Most proteins must be folded before they are
functional.
Proteins do important work
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A prion is a normal protein that has folded
incorrectly, and can cause other proteins to do
it, too. Mad cow disease is an example of a
prion infection.
BOVINE SPONGIFORM ENCEPHALOPATHY (in
cows) CREUTZFELDT-JAKOB disease (in humans)
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examples

• Sometimes biotechnology can run afoul of the laws
  of nature.

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BIOTECHNOLOGY is not new

• 2500 BC Egyptians domesticate geese
• 1800 BC Microorganisms (yeast) harnessed to
  produce beer, wine and leavened bread
• 1930-1965 US corn production up 600 due to
  development of hybrid seeds
• 1982 Insulin mass produced via biotech.
• 1990 Enzyme (chymosin) replaces rennet in cheese
  production
• 1994 Flavrsvr tomato produced
• 1999 Golden Rice introduced to prevent
  childhood blindness in developing countries

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Genetically Modified Riceexample of artificial
selection
Gene In
ß carotene
Gene Out
GMO
Wildtype
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Genetically Modified Foods

• 1 million people each year die of malnutrition
• Current goals for modification
• Improved Nutrition Profiles
• Insect Protection
• Herbicide Tolerance
• Disease Protection
• 124 Million children around the world show
  vitamin A deficiencies
• Vulnerable to infection - Mental impairment -
  Blindness
• Ingo Potrykus and Peter Beyer golden rice
• Vitamin A deficiencies can be solved by the
  addition of ß carotene (precursor for vit. A)
• ß carotene a major ingredient of carrots and some
  flowers
• Potrykus and Beyer wanted to insert the ß
  carotene gene into rice so it could become a
  vehicle to deliver vitamin A to impoverished
  people

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But how do you cut a gene out of a chromosome?
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BACTERIOPHAGE
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Restriction Enzymes

• Naturally found in bacteria
• Purpose in bacteria is to destroy foreign DNA
  (prevents infection by viruses or other bacteria)
  
• Enzymes cut DNA at specific sequence of bases
• Can be used to splice in DNA from other source
• Gene Therapy
• Use restriction enzymes to insert normal gene
  (ex nasal spray to treat cystic fibrosis)
• Tests have had mixed results video clip

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Splicing DNA with restriction enzymes
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But what if you need many copies of the gene?
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PCR

• PCR polymerase chain reaction
• Makes multiple copies of a DNA sequence
• DNA is heated, separates added primers then copy
  both strands
• Process is repeated to make many copies in a
  short time
• PCR Rap watch it here!
• http//www.youtube.com/watch?voCRJ4r0RDC4feature
  related

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Polymerase Chain Reactions

• PCR allows one section of DNA to be copied many
  times. There are many applications of PCR.
• Crime scene analysis,
• research,
• evolutionary taxonomy,
• species identification etc.

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So now that the gene is cut out of the
chromosome, where can we put it?
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Gene Therapy Treating Disease and Infertility

• Ex Vivo
• Gene is inserted into the cells that have been
  removed and then cells are returned to the body
• SCID (Severe Combined Immunodeficiency Disease)
• In Vivo
• Gene is delivered directly into the body
• Cystic fibrosis

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Transgenic plant with Fluorescent gene from
Jellyfish
Transgenic Garden peas Pesticide resistance
Transgentic Tobacco Herbicide resistant
Control Plants
Salt resistant and drought resistant tomato
Salt resistant tomato is really interesting
because the flavor of the tomato is not
affected. The salt is stored in the plants
leaves, which also desalinates the soil.
(Good for agriculture)
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Bacteria provide the restriction enzymes to cut
out genes. What else can bacteria do?
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What is a Plasmid?
Plasmid
Small circular piece of bacterial DNA. Not part
of the bacterial chromosome. Plasmid DNA
naturally carries genes for antibiotic
resistance. Why would we want to insert a gene
into a plasmid?
Chromosomal DNA
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Plasmids are tiny circular chromosomes in
bacteria
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Splicing DNA with restriction enzymes
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Final product is mix of DNA sources recombinant
DNA
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Restriction site
Plasmid DNA From Bacteria
5?
3?
G A A T T C
3?
5?
C T T A A G

• Why would we want to use the same restriction
  enzyme to
• open the plasmid AND
• isolate the gene of interest?

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Add restriction enzyme scissors
G
A A T T C
C T T A A
G
Sticky ends
A A T T C
gene of interest from another organism
G
G
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C T T A A
Using the same enzyme scissors would leave the
same bases on the sticky ends, allowing them to
bond and be sealed by DNA ligase.
Fragment cut by same restriction enzyme
G
G
A A T T
C
A A T T C
C
T T A A
G
C T T A A
G
One possible combination
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Recombinant DNA molecule
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Modeling Recombinant DNA with Paper Plasmids
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Where would this restriction enzyme cut the
plasmid and the chromosome so the gene from the
chromosome could be inserted into the plasmid?
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How do we use bacterial transformation?

• Clean up oil spills faster than natural bacteria
• Many protein products today are made through
  recombinant DNA technology
• Insulin
• Human growth factor
• Factor VIII treats hemophilia
• Tumor Necrosis Factor treats cancer
• Interferons
• Relaxin and Oxitocin child birth
• BST milk production hormone for cows
• Spider silk bullet proof vests

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Human Genome Project

• HGP
• 13 year effort
• Revealed that out of 3 billion bases only 20,500
  genes (1.5 of total DNA)
• 95 of any gene sequence is an intron
• Intergenic Sequences
• DNA sequences between genes (introns are
  sequences within genes)
• Repetitive elements, transposons, unknown
  sequences, etc.

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Unknown Sequences

• Genes comprise 1.5 of genome
• Repetitive elements are 44 of genome
• Tandem repeats
• Alu sequences
• Transposons
• The function of the rest of the genome remains a
  mystery
• However, 74 to 93 of the genome is transcribed
  just not necessarily translated

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Comparative Genomics
3 billion 2.5 billion 180 million 125 million
97 million 12 million
20,500 30,000 13,600 25,500
19,100 6,300
46 40 8
10 12 32
Mouse and human are 85 genetically identical
Chimpanzees and humans are 98 identical How
these small changes to DNA sequences and
differences in genome architecture lead to
such different organism is still not fully
understood
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Gel Electrophoresis
Anode
Electric Current

• Standard laboratory technique
• Used to separate DNA strands based on size
• Soft agar (like jello) with wells (pits) to hold
  DNA sample
• An electric current is applied
• Rate of movement is determined by size
• Larger fragments are trapped in gel matrix
• Smaller fragments move quickly through the gel
  matrix
• DNA is negatively charged so it travels towards
  the positive end

Gel
Cathode
Longermolecules
Shortermolecules
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DNA Suspect 1
350bp
100bp
200bp
DNA Suspect 2
DNA Suspect 3
375bp
250bp
Because of differences in the base sequence at
certain points, different restriction enzymes cut
at different locations on the DNA strand.
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Restriction Fragment Length Polymorphism (RFLP)
Analysis
Blood or tissue samples contain DNA from all 46
chromosomes. Incubate DNA in Test tube with
restriction enzymes scissors Every persons
DNA is different and only 5 of DNA codes for
proteins Contains many introns and non-coding
sequences which differ in the population
significantly and contain a different pattern of
cut sites. Every person will have a
different pattern after electrophoresis. DNA
fingerprint
DNA Suspect 1
DNA Suspect 3
DNA Suspect 2
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-
500
400
300
Size Base Pairs
200
100
75
50

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Size Markers
Crime Scene
Sus1
Sus3
Sus2
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Who is going to jail?
In reality the process is slightly more complex.
The total DNA is cut and then run on a gel.
However, this results in a smear of DNA down the
gel that is then stained. After staining, the
differences in the banding patterns become
apparent.
Who is the father?
Child Mom A B C
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Cloning

• Cloning
• Process which removes a diploid nucleus from a
  somatic cell and places it in a enucleate egg
• Treat with electric shock and implant into viable
  uterus

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Not all reproduction is sexual
SEXUAL 2 parents Parents are genetically
unique Offspring share traits with both parents
ASEXUAL 1 parent ALL OFFSPRING ARE GENETICALLY
IDENTICAL to the one parent. Ex cloning
A clone can be produced if the genome of a single
cell is placed in an ovum (egg) and the ovum is
stimulated to develop into a new organism that
is genetically identical to the original
organism. The sheep Dolly is a clone of its
"mother.
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CLONING
Nucleus
Remove haploid nucleus
Ovum (haploid)
Inject diploid nucleus from adult cell
Activate division