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Genetic Engineering
Chapter 13
•Recombinant DNA
•Transformation
•Biotechnology
•Gel Electrophoresis
•PCR
Biotechnology
- Any technological
application that uses
biological systems to
make or modify
products or
processes
Genetic Engineering
• Cohen and Boyer
constructed a
creature that was
part bacterium and
part frog
• Vacanti grew an ear
on a mouse
• How did they do this?
Involves Recombinant DNA
• DNA that contains DNA segments from 2
separate organisms
• Restriction enzymes are used to cut
DNA molecules only in specific places
– Cut at a specific sequence of bases called the
recognition sequence
– For example, Hind III enzyme recognizes and
cuts DNA at 5’AAGCTT3’
– Break bonds between the 3’ hydroxyl group
of 1 nucleotide and the 5’ phophate group of
the next nucleotide
• Cuts are done on both strands, are very
specific and are staggered
– The fragment ends are called sticky ends
because they can pair up with a
complementary single strand of DNA with the
help of DNA ligase
– They are able to form hydrogen bonds with
complementary sequences on other DNA
molecules
Example of a Restriction Enzymes
EcoR1 cuts at 2
different points (at
arrows)
EcoR1 cuts DNA into smaller
fragments that have sticky
ends
Sticky ends hydrogen bond to
complementary DNA with DNA ligase
• Fragments cut by same restriction enzyme
are usually inserted into a plasmid – which
is a small circle of bacterial DNA thus
creating recombinant DNA
• The recombinant DNA would then be
introduced into a bacteria hoping that the
bacterial cells would take up the
recombinant DNA
How do we know if the bacteria
contained the recombinant DNA?
By many genetic engineering
applications
1. Southern Blot
• Technique that uses a probe (specific
ordered nucleotide bases complementary
to the recombinant DNA)
– Extract the DNA
– Cut up the DNA
– Separate the fragments
– Transfer the fragments to a filter sheet
– Remove filter and attach probes
– Create autoradiograph
autoradiograph
2. Gel Electrophoresis
• Technique which
would separate
DNA fragments
across an electric
field
• We are doing this
is in AP Lab 9
Gel Electrophoresis – how does
it work?
• DNA is negative charged due to the
phosphate groups
• So DNA will be put on an electric field and
will move toward the positive end
• Small DNA molecules can move through
the gel faster than larger ones
• Gives us information about DNA (size of
fragment, number of fragment)
3. RFLP’s
• Restriction fragment
length polymorphism
• Technique used to
determine how
closely related the
length of DNA
fragments are
• Used in forensics to
identify a person
• Used to settle
disputed parentage
4. PCR
• Polymerase chain
reaction
• Technique of
amplifying a tiny
sample of DNA in
a few hours
• Used to duplicate
crime scene DNA
etc.
13.2
• One goal of recombinant DNA technology
is to clone
– Produce many identical copies of a particular
DNA sequence
– Like PCR does!
Transformation
• Inserting recombinant DNA into a host cell
• The cell or organism that is the host
containing the inserted recombinant DNA
is called transgenic.
Insulin
• Essential for glucose uptake into cells
• Is a good illustration of a medical application to biotechnology
• People with type I diabetes cannot make this hormone and must
receive insulin injections
• In the past it was made by cattle and pigs, now from E. Coli!
• It is a protein of 51 amino acids and is made up of 2 polypeptide
chains
• Cattle DNA differs from human DNA by 3 amino acids and pigs just
by 1 amino acid (in the amino acid sequence of insulin)
• This difference causes immune responses in diabetics, so these
patients need insulin from humans
– Harvesting the protein from deceased humans does not make enough
• So Recombinant DNA biotechnology solves the problem!
Pharming
• Another way of making medically useful products in large amounts
• The production of pharmaceuticals in farm animals or plants
Example
• Human Growth Protein, made in pituitary gland
• People with deficiencies in this are short etc.
• In the past they were treated with protein isolated from the pituitary glands
of dead people
• Supply was too limited and a demand for it resulted
• So Recombinant DNA technology was used – bacteria can make the
protein, but it is expensive ($30,000 a year)
• So now a transgenic cow is used that secretes the human growth hormone
is the milk.
• Only 15 cows are needed the meet the world wide demand of dwarfism
DNA Manipulation Examples
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Larger seeds in plants
Low fat milk
Plants that are resistant to disease
Plants that make their own insecticides
Corn with high amounts of B-carotene
Crops that adapt to the environment
And more!