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Chapter 20
DNA Technology
P 365
Animation
Plasmid
Recombinant DNA
Ligase seals
backbone
Only some bacteria get
a plamid
Antibiotic added
to screen out
those without
plasmid
Only one
plasmid has the
FROG gene
Frog rRNA
labelled with
radioactive
tracer is used to
find which
plasmid has the
frog gene
Bacteria with frog gene can
now be cloned
Penicillin link
The plasmids are reinserted into the bacteria, but only
some take up the plasmid -they must be SCREENED OUT
DNA probe (with
radioactive tag)
complementary to
frog gene
Animation
P 367
Plasmid has 2 genes
Bacteria that
take up the
plasmid will gain
antibiotic
resistance
Blue colonies
have the lactase
gene
White colonies
DON’T have the
lactase gene but have the
human gene
Bacteria that
have also taken
up the Human
gene will lose
the lactase gene
Cloning Animation
Another cloning animation
Animation of removing
introns to put eukaryotic
gene in prokaryote
Pearson Lab 6A simulation
Turn in Lab Quiz 1 tomorrow.
Five Stages in Genetic
Engineering
1.
2.
3.
4.
5.
•
•
Isolate and Cleave DNA
Produce Recombinant DNA
Introduction of Vector into target cell
Clone Cells
Screen Target Cells
(Clone the screened cell once the target cell is
chosen)
Usually genetically modified bacteria are
crippled so that it cannot survive outside lab
P 369
Eukaryotic Genes
must be modified
to work in bacteria
Expression Vector
is created
1. A Promotor must
be inserted
2. Introns must be
removed
Genes can be
found using
tagged cDNA
All the genes of
an organism
represents a
Genomic
Library and may
be stored in a
series of
vectors such as
viruses of
bacteria
DNA Libraries Link
Plucky is an albino
Xenopus laevis frog
expressing green
fluorescent protein
(GFP) in her eye. GFP is
a jellyfish protein that
fluoresces bright green
when illuminated by
blue light.
Pioneering genetic
engineering on mammals
have been been done at
UH
Cloning 101 link
THE FIRST MOUSE CLONES
The clones (two brown mice at bottom) are genetic duplicates of the
mouse at top right, which donated its cumulous cells. They are the
result of a technique perfected at the University of Hawaii in 1998
WILBUR WANNABES
The first litter of cloned pigs, born in 2000 in Virginia, demonstrate that
cloning could be used to generate organs for human transplant in the
near future
Dr. Severino Antinori, an Italian
embryologist, fires up the press in
2001 after announcing plans to clone
the first human to help infertile
couples have children. He claimed
one of his patients was carrying a
clone, but he failed to confirm his
tale or produce the child. His
comments launched a debate over the
ethics of cloning human beings;
countries like Britain and South
Korea have since made it illegal to
clone people, while the U.S.
Congress has yet to ban the process.
Not to be outdone, Chinese researchers are perfecting cloning
techniques in the hope of using the procedure to preserve the
country's beloved panda species. For practice, they began with
more common species, including goats like Yangyang (above).
Cloning remains a tricky process; only 2%-5% of the eggs that start
out as clones develop into live animals. The good news is that once
they survive past the first year, clones like Yangyang, celebrating
her sixth birthday, are relatively healthy.
Stop Three-Parent Babies
Scientists: Regulate Fertility Clinics To Prevent Babies with New Genes
By Robin Eisner
N E W Y O R K, May 18 — Scientists are calling for the immediate regulation of
fertility clinics to prevent the birth of any future gene-altered babies, the first of
which was reported earlier this year.
STORY HIGHLIGHTS
Fertility Method Creates Gene-Altered Babies Extra Genes From Mitochondria
Social and Safety Consequences of Technology
In March, a team of fertility specialists at the Institute for Reproductive Medicine and
Science of St. Barnabas, in West Orange, N.J., reported "the first case of human …
genetic modification resulting in normal healthy children."
Fertility Method Creates Gene-Altered Babies
The group used a method that extracted cellular material from a donor woman's egg
cell and transferred it into an infertile woman's egg. This material allowed the
woman's egg to become fertile.
The donor egg contained DNA from mitochondria, little organs inside the cell that
create the energy to do life's work. The group believes that problems with the
mitochondria prevented the infertile women from becoming pregnant.
Mitochondria contain only about 0.03 percent of a cell's DNA, but that's enough that
they can make copies of themselves when the cells divide. The other 99.97 percent of
a cell's DNA comes from the nucleus and the 23 pairs of chromosomes.
The group says that transferring this mitochondrial DNA into the recipient eggs
resulted in the birth of 30 babies, the first of which was born in 1997.
Review Random
Fertilization
Geneticists had taken
the luciferase gene
from a firefly and
inserted it into a
tobacco plant. This
meant that when the
plant was fed with
luciferin the result was
a plant that glows in
the dark!
Animation
Link to PCR Animation on Web
Different people have
different DNA
-DNA when cut by
enzymes will leave
different size fragments
-which will separate into
different electrophoresis
patterns
-a DNA fingerprint
If there is only a small
sample of DNA
available- more copies
can be made by PCR polymerase chain
reaction (p371)
Link to DNA
Fingerprint Lab
P 373
Differences in DNA sequences on homologous
chromosomes result in different restriction fragment
length patterns RFLP
- these may be sorted by length using gel
electrophoresis
Specific genes (or fragments), how many places they show up plus the DNA fragments
that they can be found is detected using Southern Blotting
1-Cut
2-Separate
3-Move fragment to
permanent substrate
4-Heat/Tag
Animation
Different restriction fragments are found
at different frequencies within different
people
For example fragment A
may be found in 50% of the
population, fragment B
might be found in 10% of
the population and
fragment C might be found
in 3% of the population
RFLP = restriction fragment length
polymorphism
• If a person has fragment A, B and C in his
DNA fingerprint and the fragments are
found in these percentages in the population
A= 50%, B= 10% and C=3%
What is the chance that someone else has the same 3
fragments in their fingerprint?
RFLP analysis identifies the presence of a specific gene by looking
for an associated RFLP marker (recognition site) near the allele.
Animation
•ANSWER= A x B x C = .5 x .10 x .03 = .0015 or
.15% or 1 out of 667 people
•Testing more fragments gives a smaller %
Certain restriction fragments can be
looked for in a person’s fingerprint
by adding a radioactive or dye
labeled DNA probe that is
complementary to the DNA of the
fragment
Why is a DNA fingerprint NOT the same as a real fingerprint?
One to one relationship
Statistical relationship
OJ Simpson Trial
• Odds of seeing 3 albino deer at the
same time:
85 million to 1
• Odds of the blood on the glove not
being from R. Goldman, N. BrownSimpson, and O.J. Simpson:
21.5 billion to 1
Pearson Lab 6B Electrophoresis simulation
Turn in Lab Quiz 2 tomorrow.
Mapping Genomes
3 steps
• Genetic (linkage) mapping (this was covered
earlier –remember in the fly lab, the black body
with vestigial wings genes were mapped using
crossing over frequencies)
• Physical mapping
• DNA sequencing
Matching overlapping sequences allow scientist to put all the
fragments in order
Scientists have now sequenced the entire Human Genome
opening up the Human Genetic Library for research
DNA Sequencing Link
Sequencing of DNA
is accomplished
by copying one side one base
Figure
13-7 DNA Sequencing
at a Section
time using
a modified base with a dye molecule.
13-2
Dye molecules
replace -OH
group – stops
replication
DNA Microarray Assay of Gene Expression
Levels. Genetic testing (genetic screening)
allows the genetic diagnosis of vulnerabilities
to inherited diseases, and can also be used to
determine a person's ancestry
animation
Slide 36Animation
link
Ti plasmids in
bacteria can cause
tumors in plants
If the tumor causing gene is removed
from the plasmid and a useful gene
spliced in - the Ti plasmid can be used as
a VECTOR to move genes into plants
Ti plasmid uses Agrobacterium tumefaciens to
transduce its genetic material to plants
Cannot be used on grain plants
Practical Uses of DNA Technology
Diagnosis of disease
Human gene therapy
Pharmaceutical products
(vaccines)
Forensics
Animal husbandry (transgenic
organisms)
Genetic engineering in plants
Ethical concerns?
15.9
A genetically modified tobacco
plant designed to resist a virus
Enzymes that
eat holes in the
gut of the
caterpillar
inserted into the
plant.
Nodules in Legume plant
Nitrogen fixing bacteria live in the nodules and provide the plant with a source of nitrogen
Nitrogen is needed to make proteins and DNA.
Scientists are trying to insert the enzymes to fix nitrogen into other plants to save on
costly nitrogen fertilizers.
Grain and Cereal
Plants cannot use
the Ti plasmid as a
vector, but genes
can be inserted
using a DNA gun
A DNA gun can be used to shoot genes into cells or cell
parts like chloroplasts
Growth
hormones
can increase
yield and in
cows,
increase milk
production
Genes for
Human INSULIN
might be
inserted into
plasmids
Bacteria can
now produce
Human insulin
Proteins or
ANTIGENS on
the surface
AIDS is an virus
containing RNA
-the information for
surface Antigens is
located here
AIDS
antigen
gene
Humans will
make
ANTIBODIES to
attack the AIDS
ANTIGENS
Cowpox
viruses can be
made with
AIDS proteins
(ANTIGENS)
on its surface
Vaccine animation
Inactivated cold viruses
can be used as a
VECTOR to move the
genes for ANTIGENS from
other viruses into a
person.
For example this virus
might carry genes for the
herpes surface protein. If
this was injected into a
person, he would make
ANTIBODIES for the
herpes proteins -thus
giving him immunity to
herpes.
ADA: The First Gene Therapy
Trial
• A four-year old girl became the first gene therapy patient
on September 14, 1990 at the NIH Clinical Center. She has
adenosine deaminase (ADA) deficiency, a genetic disease
which leaves her defenseless against infections (SCIDS).
White blood cells were taken from her, and the normal
genes for making adenosine deaminase were inserted into
them. The corrected cells were reinjected into her. Dr. W.
French Anderson helped develop this landmark clinical
trial when he worked at the National Heart, Lung, and
Blood Institute
Cystic Fibrosis
• CF causes the body to produce thick, sticky
mucus that clogs the lungs, leads to
infection, and blocks the pancreas, which
stops digestive enzymes from reaching the
intestine where they are required in order to
digest food.
Cystic Fibrosis: A Single Gene
Disease
• Mutations in a single gene - the Cystic Fibrosis
Transmembrane Regulator (CFTR) gene - causes
CF. (autosomal recessive)
• In normal cells, the CFTR protein acts as a
channel that allows cells to release chloride and
other ions. But in people with CF, this protein is
defective and the cells do not release the chloride.
The result is an improper salt balance in the cells
and thick, sticky mucus.
Gene Therapy Research Offers
Promise of a Cure for Cystic Fibrosis
In 1993, the first
experimental gene therapy
treatment was given to a
patient with CF. Researchers
modified a common cold
virus to act as a delivery
vehicle - or "vector"carrying the normal genes to
the CFTR cells in the
airways of the lung.
Leber congenital amaurosis (LCA) is an inherited retinal
disease that causes severe visual impairment in infancy or
early childhood. Current research on a gene transfer therapy
may offer hope to people with a form of this disease
Genetic Engineering may have
environmental risks
Can you name some?
Altering a person’s genes
to combat disease is called
Gene Therapy
TIL Tumor
Infiltrating
Lymphocytes
have the ability
to find and slow
the growth of
tumors
Changing only the genes in body
cells like lymphocytes is called
somatic cell gene therapy
By genetically adding
the TNF gene to these
cell will increase their
effectiveness at
destroying tumor cells
Chapter 20
DNA Technology
and Genomics
•
The principal problem with inserting an
unmodified mammalian gene into the bacterial
chromosome, and then getting that gene
expressed, is that
A. prokaryotes use a different genetic code from that
of eukaryotes.
B. bacteria translate polycistronic messages only.
C. bacteria cannot remove eukaryotic introns.
D. bacterial RNA polymerase cannot make RNA
complementary to mammalian DNA.
E. bacterial DNA is not found in a membraneenclosed nucleus and is therefore incompatible
with mammalian DNA.
• Which of the following statements is consistent
with the results below? *
A. B is the child of
A and C.
B. C is the child of
A and B.
C. D is the child of
B and C.
D. A is the child of
B and C.
E. A is the child of
C and D.
•
Which of the following statements is most likely true?
A. D is the child of
A and C.
B. D is the child of
A and B.
C. D is the child of
B and C.
D. A is the child of
C and D.
E. B is the child of
A and C.
•
Which of the following are probably siblings?
A.
B.
C.
D.
E.
A and B
A and C
A and D
C and D
B and D
• The segment of DNA shown in
the figure below has restriction
sites I and II, which create
restriction fragments A, B, and
C. Which of the gels produced
by electrophoresis shown
below would represent the
separation and identity of these
fragments?
•
This restriction fragment
contains a gene whose
recessive allele is lethal. The
normal allele has restriction
sites for the restriction
enzyme PSTI at sites I and II.
The recessive allele lacks
restriction site I. An
individual who had a sister
with the lethal trait is being
tested to determine if he is a
carrier of that allele. Indicate
which of these band patterns
would be produced on a gel if
he is a carrier (heterozygous
for the gene)?