Download Ch. 13 Genetic Engineering

Ch. 13 Genetic Engineering
Ch. 13 Outline

13-1: Changing the Living World
Selective Breeding
 Increasing Variation
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13-2: Manipulating DNA
The Tools of Molecular Biology
 Using the DNA Sequence

Ch. 13 Outline
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13-3: Cell Transformation
Transforming Bacteria
 Transforming Plant Cells
 Transforming Animal Cells
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13-4: Applications of Genetic Engineering
Transgenic Organisms
 Cloning

Selective Breeding
 Humans
use selective breeding,
which takes advantage of naturally
occurring genetic variation in plants,
animals, and other organisms, to
pass desired traits on to the next
generation of organisms
Selective Breeding
 Nearly
all domestic animals -including horses, cats, and farm
animals – and most crop plants
have been produced by selective
breeding
Hybridization
 Louis
Burbank was the greatest
selective breeder of all time. He
developed the disease-resistant
potato and more than 800
varieties of plants.
 Louis
Burbank used the technique
of hybridization and bred
dissimilar individuals to combine
the best traits of both parents.
 The
hybrids produced by these
crosses were hardier than their
parents
Inbreeding
 To
maintain the desired
characteristics of a line of organisms,
breeders often use the technique of
inbreeding.
 Inbreeding
is the continued breeding
of individuals with similar
characteristics
Increasing Variation
 In
order for selective breeding to be
successful, there must be a lot of genetic
variation in the population
 Breeders
increase the genetic variation
in a population by inducing mutations,
which are the ultimate source of genetic
variability
Increasing Variation
 Breeders
increase the mutation rate by
using radiation and chemicals
Producing New Kinds of Bacteria and Plants

Using radiation or chemicals, scientists
have been able to develop hundreds of
useful bacteria strains

Drugs that prevent chromosomal separation
during meiosis have been used to produce
plants that have many sets of
chromosomes.

The hybrid plants are polyploid.
What is genetic engineering?

In 1973, Mr. Cohen and Mr. Boyer
conducted an experiment on the DNA of
an American frog.
What is genetic engineering?

They found and isolated the gene that
codes for ribosomal RNA (rRNA) in the
DNA of the frog. They removed that gene
from the frog and inserted it into some E.
Coli Bacteria.
What Happened?

During transcription, the bacteria then
produced the frog RNA!

Genetic Engineering: the process of
manipulating (moving) genes for
practical purposes (useful)

Recombinant DNA: DNA made from 2 or
more organisms that are different.
The Basic Steps of Genetic Engineering
1.
Cutting the DNA:


Restriction Enzymes: bacterial enzymes that
recognize and bind to specific short
sequences of DNA, and then cut the DNA
between specific nucleotides within the
sequences.
Vector: agent used to carry the gene of
interest – usually plasmids

Plasmid: the circular DNA molecules that
replicate
The Basic Steps to Genetic Engineering
2.
Making Recombinant DNA


3.
DNA fragments of interest (that have already
been cut) are combined with the vector.
DNA ligase – the enzyme bonds the 2 ends of
the fragments to the vectors.
Cloning

Gene cloning: the process of making many
copies of a gene

Bacteria reproduce by binary fission
The Basic Steps to Genetic
Engineering
4.
Screening


Cells that have received the gene of
interest are separated out.
Those cells then continue to produce the
protein coded for by the gene
Cutting DNA & Making Recombinant DNA

How Restriction enzymes work:



The Enzymes recognize specific sequences on
Human and Bacterial Plasmids
The Enzymes cut the strands.
The cut produces DNA fragments with short strands
on each end that are complementary to each other


“Sticky Ends”
Both the human DNA and the Plasmid “Open Up”
with the same sticky ends remaining

They Bind Together
Diagram
Recognition
sequences
DNA sequence
Recognition sequences
DNA sequence
Restriction enzyme EcoRI cuts
the DNA into fragments.
Sticky end
Confirmation of a Cloned Gene


One method used identify a specific
gene is called a Southern Blot
Steps:
1.
2.
Cut DNA from bacteria with restriction
enzymes.
DNA fragments are separated by a gel
soaked in a chemical solution.

Gel electrophoresis – uses an electric field
within a gel to separate molecules by their size
Confirmation of a Cloned Gene
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Negatively charged DNA is put into these
wells.


They are attracted to the positive pole from
the electric field.
The Smallest DNA fragments move the
fastest
Gel Electrophoresis
DNA plus
restriction enzyme
Power source
Longer
fragments
Mixture of
DNA
fragments
Gel
Shorter
fragments
Confirmation of a Cloned Gene
3.
The DNA separated is then transferred to a
filter paper (blotted) and a probe solution is
added.

4.
Probes: radioactive RNA or single-stranded
DNA pieces that are complementary to the
gene of interest
Only DNA fragments complementary to the
probe will form and bind bands
Confirmation of Cloned Genes
 Why do this?
 Bacterial
colonies can be used to
produce large quantities of the protein
(used to study or make drugs)
Confirmation of Cloned Genes
 When
a bacteria or other cell takes
in a foreign piece of DNA such as a
plasmid, the process is called
transformation
 If
transformation is successful, the
recombinant DNA is integrated into
one of the chromosomes of the cell.
Improving Crops
 Genetic
engineers can add favorable
characteristics to a plant
 Plants
become resistant to insects
(no longer need pesticides); resistant
to weed killer (so crops won’t die, but
weeds will); improved nutrition – rice +
corn
Plant Transformation
Gene to be
transferred
Inside plant cell,
Agrobacterium
inserts part of its
DNA into host cell
chromosome
Agrobacterium
tumefaciens
Cellular
DNA
Recombinant
plasmid
Plant cell
colonies
Complete plant is
generated from
transformed cell
Transformed bacteria introduce
plasmids into plant cells
Animal Farming

Growth hormones is given to cows to
produce more milk

Human genes are added to farm animals in
order to have human proteins in their milk
 The Human proteins are extracted from
milk and sold to pharmacy companies.
Useful
for complex proteins that can’t be
made in bacteria
Creating HGH
Recombinant
DNA
Gene for human
growth hormone
Gene for human
growth hormone
Human Cell
Sticky
ends
Bacterial Cell
Bacterial
chromosome
Plasmid
DNA
recombination
DNA
insertion
Bacterial cell for containing gene
for human growth hormone
Animal Farming
 Transgenic
animals: Animals that
have foreign DNA in their cells
 Cloning
of animals is another way to
make large quantities of a certain
protein.
Animal Farming

Transgenic animals:
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How it works: an intact nucleus from an
embryonic cell (whose DNA has recombined
with a human gene) is placed into an egg
whose nucleus has been removed.

The “new” egg is then placed into the uterus
of an animal.
Cloning Animals

A clone is a member of a population of
genetically identical cells produced from a
single cell

How it works: an intact nucleus from a cell is
removed
Cloning Animals
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The nucleus is fused with a egg cell (whose
nucleus has been removed) taken from
another adult

The fused cell begins to divide and the
embryo is placed in the uterus of a foster
mother.

The “new” egg is then develops normally.
Cloning
A donor cell is taken from a
sheep’s udder.
Donor Nucleus
Egg Cell
These two cells are fused using an
electric shock.
Fused Cell
The nucleus of the egg cell is removed.
The fused cell begins dividing
normally.
Cloned Lamb
An egg cell is taken from an
adult female sheep.
The embryo is placed in the uterus of
a foster mother.
Embryo
The embryo develops normally into a lamb—
Dolly
Foster Mother
Genetically Engineered Drugs and Vaccines

Today, many pharmaceutical companies
around the world produce important
proteins using genetic engineering.

Vaccine: a solution containing all or part of a
harmless version of a pathogen; used to
prevent viral diseases (don’t respond to
drugs)

Many vaccines are made using genetic
engineering
DNA Fingerprinting
 DNA
fingerprinting:
a
pattern of dark bands on
photographic film
 an
individuals’ DNA restriction
fragments are separated by gel
electrophoresis, probed, and exposed
to X-ray film.
DNA Fingerprinting
 DNA
fingerprints can be used to
 establish
 identify
paternity
genetic disorders
 forensics
(scientific study of cause of
injury or death in criminal activity)
Three paternity cases tested
with DNA purified from blood
samples. 1: mother; 2: child; 3:
alleged father; 4: child +
alleged father. M: markers. 3
µg of DNA was loaded per
lane. Outcome: A: alleged
father excluded; B & C:
alleged father confirmed.
(Data kindly provided by J.
James, Gene Proof
Technologies, Nashville, TN,
USA.)