Download Recombinant DNA and Genetic Engineering

Golden Rice
or Frankenfood?
• Scientists transferred
daffodil genes into rice
• Rice with beta-carotene
may help prevent vitamin
A deficiencies
• Opponents fear
unforeseen consequences
of creating genetically
modified organisms
Genetic Changes
• Humans have changed the genetics of
other species for thousands of years
– Artificial selection of plants and animals
• Natural processes also at work
– Mutation, crossing over
Genetic Engineering
• Genes are isolated, modified, and
inserted into an organism
• Made possible by recombinant DNA
technology
– Cut up DNA and recombine pieces
– Amplify modified pieces
Discovery of Restriction Enzymes
• Hamilton Smith studied how bacterium
Haemophilus influenzae cut invading
viral DNA
• Discovered that bacteria have an
enzyme that chops up viral DNA
Specific Cuts
• Restriction enzymes cut DNA at a
specific nucleotide sequence
• Cuts leave “sticky ends” on DNA
fragments that pair with matching cuts
Making Recombinant DNA
5’
G
3’
C T T A A
A A T T C
G
one DNA fragment
another DNA fragment
5’
G
A A T T C
3’
3’
C
T T A A
5’
G
Making Recombinant DNA
nick
5’
G A A T T C
3’
3’
C
5’
T T A A
G
nick
DNA ligase action
G A A T T C
C T T A A G
Using Plasmids
• Plasmid: a small circle of bacterial DNA
• Foreign DNA inserted into plasmid
– is replicated and distributed along with plasmid
• Plasmid becomes a cloning vector
– delivers DNA into another cell
Cloning Vectors
Using Plasmids
a A restriction enzyme
cuts a specific base
sequence everywhere
it occurs in DNA.
c The same enzyme cuts
the same sequence in
plasmid DNA.
b The DNA fragments
have sticky ends.
e The DNA
fragments and
plasmid
DNA are
mixed with
DNA ligase.
f The result? A collection
of recombinant plasmids
that incorporate foreign
DNA fragments.
d The plasmid DNA
also has sticky ends
g Host cells
that can divide
rapidly take up
the recombinant
plasmids.
Fig. 11-3, p.164
Gene Libraries
• Cells that host fragments of cloned DNA
– Genomic library
Where am I?
123 Devilbiss
Biology 101
SU
US
Earth
47%
35%
12%
6%
rt
h
Ea
S
U
SU
10
og
y
io
l
B
3
De
v
ilb
is
1
s
0%
12
1.
2.
3.
4.
5.
DNA Sequencing
• Order of
fluorescent
bands indicates
DNA sequence
Polymerase Chain Reaction
• Sequence to be copied is heated
• Primers are added and bind to ends of
single strands
• DNA polymerase uses free nucleotides
to create complementary strands
• Doubles number of copies of DNA
Polymerase
Chain Reaction
double-stranded
DNA to copy
DNA heated to
90°– 94°C
primers added to
base-pair with
ends
mixture cooled;
base-pairing of
primers and ends
of DNA strands
DNA polymerases
assemble new
DNA strands
Polymerase
Chain Reaction
mixture heated
again; makes all DNA
fragments unwind
mixture cooled; basepairing between
primers and ends of
single DNA strands
DNA polymerase
action again
doubles number of
identical DNA
fragments
DNA Fingerprints
• Unique array of DNA fragments
• Inherited from parents in Mendelian
fashion
• Even full siblings can be distinguished
from one another by this technique
Analyzing DNA Fingerprints
• DNA is separated by gel electrophoresis
• Pattern of bands is used to
– Identify or rule out criminal suspects
– Identify bodies
– Determine paternity
A DNA Fingerprint
DNA Sequence Data
• Evolutionary trends
• Potential therapies for genetic diseases
• Data bases using computer technology
Genetics in Practice
• Genetic engineering is used to change
genetic makeup of organisms
– Genes transferred between species
(Transgenic organisms)
– Genes modified and reinserted into same
species
Transgenic Plants
• Contain DNA from another species
• New genes make crop plants less
vulnerable to disease and pests
• Use bacterial cloning vectors to insert
foreign gene (Ti plasmid)
Transgenic Cotton
• Resistant to herbicides
• Insecticide gene
Genetically Engineered Bacteria
• Produce medically valuable proteins
• Breakdown environmental contaminants
• Designed to survive only under narrow
conditions
Transgenic Animals
• Early experiments in mice
– Injection of rat gene corrected a growthhormone deficiency
– Injection of human growth-hormone gene
produced giant mice
• Human genes are now routinely
transferred into animals to produce
human proteins for use as drugs
Genetically Modified Animals
Gene Therapy For SCID-X1
• Designed to cure “bubble babies”
– Immune system can’t fight infection
Gene Therapy For SCID-X1
• Retrovirus inserts normal allele into
cultured stem cells
• Modified stem cells returned to child’s
bone marrow
• Successfully created immune function
• Also caused leukemia in some children
Who Gets Enhanced?
• Eugenic engineering
– Selecting for “desirable” human traits
• How should we use gene therapy?
• Who should decide what genetic traits
can or should be altered?
Xenotransplantation
• Human organs are in short supply
• Pig organs are similar, but human body
rejects them as foreign
• Scientists work to knockout pig genes
that trigger rejection
• Can interspecies transplants introduce
new diseases to humans?
“Frankenfood”
• Genetically engineered foods are
widespread in the US
• Cut costs, reduce herbicide use, enhance
yields
• What effect will they have on humans
and ecosystems?
Transgenic Mouse
• With genes
for
fluorescent
protein