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Genetic Technology
DNA Fingerprinting
• We can compare DNA from
two people by running each
person’s DNA on a gel.
(GEL ELECTROPHORESIS)
• It is not an actual fingerprint,
but unique in that every
person has different DNA
sequences (except for
identical twins).
• Therefore, they will have
different band patterns in a
gel.
Applications of DNA Fingerprinting
• Determine paternity
– The bands from a gel electrophoresis of a child’s
DNA will match with the mother’s or father’s
•Crime Scene applications
•It is hard to find an intact fingerprint at a crime scene; it is
easier to find bodily fluids, hair and skin cells left behind
•Personal Identification, for example: US Military
Tobacco Plant
Firefly
How are restriction enzymes
useful?
• We can cut whole genes
from an organism (ex:
protein that produces light
from firefly).
• We can insert that gene
into a plasmid (bacterial
DNA).
• We can then put that
whole bacterium into a
tobacco plant.
• The bacteria replicates
and produces the protein
inside the plant. The plant
expresses the protein.
Plant glows like a firefly.
Spider Silk—is the strongest
material known to man!
Goat—Produces milk
The female genetically modified goats can act like silk
protein factories when they give birth and start
producing milk.
How in the world did that happen?!
First you find the gene that
you want to transfer over
Then you must cut that gene out of the parent DNA
The area where you want to put the DNA
in the other organism has to be found
Cut the new DNA so you can insert into vector
Gene put into new organism
GGAATTCCTTAAGTCAACCGCTTAAGG
1. Gene you want.
Makes Human insulin
GGAATTCCTTAAGTCAACCGCTTAAGG
2. Cut out by restriction enzymes
4. Insert Insulin Gene here!
GTACTGACCCTTGGTA
AGAGTACGTTTGT
3. DNA inside
vector cut with
restriction enzymes
5. Bacteria produces
insulin !!!
What is a vector?
A vector is a virus that has been re-engineered
OR a plasmid, which is a ring of DNA found in bacteria
Transgene
Plasmid
Transgene
Virus
Bacteria act as vectors
Plasmid (reproduces
independently of
chromosome)
Bacterial chromosome
So why don’t we make all kinds of new animals and plants?
We don’t know how it will affect our environment
What other effects will it have besides the one intended
They can mutate
Examples of genetically modified organisms that we have
now………
Sterile male crop pests
Plants that have an insecticide in them
What are Restriction Enzymes?
Remember DNA structure??
Base Pair
Nitrogenous Bases
Restriction enzymes are proteins that cleave (cut)
DNA molecules at specific nucleotide sequences
(A,T,C, and G).
Example:
HobI cleaves at
the sequence
CAATTG
• Each restriction enzyme
cleaves DNA at a different
sequence.
• Enzyme recognition sites are
usually 4 to 6 base pairs in
length.
• The fragments created can be
separated by gel
electrophoresis.
• Restriction enzymes are taken
from a wide variety of bacteria
and are thought to be part of
the cell's defenses.
• The shorter the recognition
sequence, the greater the
number of fragments
generated.
Restriction
Enzyme
Recognition
Site
AccII
CG↓ CG
AluI
AG↓ CT
AvaI
C↓TCGAG
BamHI
G↓ GATCC
BglII
A↓ GATCT
DraI
TTT↓ AAA
EcoRI
G↓ AATTC
HindIII
A↓ AGCTT
KpnI
GGTAC↓C
PstI
CTGCA↓ G
SalI
G↓ TCGAC
SmaI
CCC↓ GGG
Sau3
A ↓ GATC
TaqI
T↓ CGA
XbaI
T↓CTAGA
(Restriction Enzyme)
(Restriction Enzyme)
Recognition Site:
GAAT T C
GATATC
Blunt or sticky
ends?
Sticky
Blunt
TRANSGENIC
ORGANISMS----
Transgenic Organisms
What are they?
Organisms that carry genes from another species
The first transgenic organisms were bacteria
First transgenic animal happened in 1975
A mouse carried an ape gene
+
Transgenic organisms
• piggyBac gene from coral inserted into mice.
Mice express the red fluorescent protein coded
for by this gene.
• Also: medicine and spider silk is produced in
goat milk. Spider silk could be used to make
bulletproof vests and sutures.
Jellyfish gene makes pigs look
yellow.
Gene Therapy
The treatment of certain disorders, especially genetic
disorders, by introducing specific engineered genes into
a patient's cells
Several methods can be used when treating a genetic disorder:
A normal gene may be inserted into a nonspecific location within the
genome to replace a nonfunctional gene. This approach is most
common.
An abnormal gene could be swapped for a normal gene.
The abnormal gene could be repaired, which returns the gene to
its normal function.
The regulation (the degree to which a gene is turned on or off) of a
particular gene could be altered
So how do we do this?
A vector is used to deliver the DNA needed to fix the problem into
the target cells……the ones that need to be fixed.
Just like in transgenic organisms the vector infects the cells and delivers
the DNA into the cell to be put into the target cell’s DNA
This new DNA changes the target cells so that they are now normal.
What kind of disorders are we talking about here?
Hemophilia
Huntington’s Disease
Sickle Cell Anemia
Cystic
Fibrosis
Hemophilia is a perfect candidate
for gene therapy—it is caused by
mutations to a single gene.
This could replace the bad
gene with a normal gene.
A vector is a method of
transportation for the gene.
Most vectors are harmless
viruses.
Lab animals have been used
to test this method and
they have been cured of
hemophilia!!!!
GENE THERAPY
NOT APPROVED BY
THE FDA YET !!!!!!
GENE THERAPY IN
CYSTIC
FIBROSIS
These lung cells from a cystic fibrosis
patient have been infected with the
evolved virus carrying a correct copy
of the CFTR gene. The cells with a
green interior are expressing high
levels of normal CFTR protein, which
is a chloride ion transporter that is
defective in CF patients because of a
mutation in the CFTR gene. The
chloride ion transport of these cells
was completely repaired with the
virus. (Schaffer lab/UC Berkeley
Mark Origer described last night how he was made well enough to attend his
daughter's wedding after doctors carried out pioneering gene therapy in which
genetically altered versions of his blood cells cleared his body of tumours.
Mr Origer, 53, the head of the US post office in Lake Mills, Wisconsin, was
diagnosed with melanoma, the most aggressive form of skin cancer, in 1999.
His wife, a nurse, had noticed that a mole on his back looked similar to a
melanoma shown on a television program. "I was unaware of it," he said.
A dermatologist removed the mole. When it tested positive for melanoma, he
also removed more surrounding tissue.
A cyst subsequently developed at the same site in 2002 and it was found to
contain malignant melanoma cells. Despite many treatments being tried, the
cancer spread relentlessly. First it reached the lymph nodes under his left arm
and, then in June 2004, his liver.
Among the treatments he tried were interferon, interleukin 2, a melanoma
vaccine and surgery. "There were emotional highs and lows, as we tried
something new and then there was a recurrence," he said.
In December 2004, he received the new gene therapy from Dr Steven
Rosenberg's team at the National Cancer Institute in Bethesda, part of the
US government's powerhouse of medical research, the National Institutes of
Health.
In earlier work by Dr Rosenberg's team, white blood cells were removed from
patients with advanced melanoma, tumour-killing cells isolated, multiplied in the
lab and then reintroduced after other white blood cells were removed by
chemotherapy. This showed some success, getting tumour regression in half of
the patients.
This time, the team also genetically altered Mr Origer's white blood cells to
recognise and attack cancer cells, making them more effective cancer-fighting
cells.
To do this, they drew a small sample of his blood that contained normal white
blood cells — lymphocytes — and infected the white cells with a retrovirus
designed to deliver genes.
The virus inserted DNA that enabled the white blood cells to make proteins,
called T cell receptors, which recognise and bind to certain molecules found on
tumour cells, so they were better able to destroy the cancer cells.
Mr Origer – who lives on a farm in Watertown and has two daughters, aged 25
and 16, and a son, aged 23 – was discharged at the end of that December and
hoped, most of all, to survive long enough for his eldest daughter's wedding.
Although the chemotherapy used to clear the way for billions of his genetically
altered white cells had made him feel fatigued, he soon bounced back. In
January, a scan showed that his tumours had shrunk by about half. His doctors
broke into wide grins as they reported the effects.
In September of last year, his daughter Katie was married and Mr Origer
was able to give the bride away. "She wanted me to be there for her, and she
wanted me to be there for me. There was a lot of concern from all my
children." By the time he walked his daughter down the aisle, only a small
spot remained visible in his liver, one that surgeons at the NCI removed in
October as part of the protocol of the trial.
Study of the "very small" spot revealed that a few cancerous cells remained.
On Monday and Tuesday of this week he was given his three-monthly
assessment by Dr Rosenberg, which involved a scan. "I'm clean," said Mr
Origer. "Perfectly clean. There is nothing showing up on the CT scans. I'm
thrilled."
Mr Origer, who has had no contact with any other participants in the trial,
will continue to go back to Bethesda for follow-up scans. "Naturally, I feel a
tremendous indebtedness to Dr Rosenberg," he said. "I know how fortunate I
am to have gone through this and responded to this. Not everybody's that
lucky."
He said that, despite his extraordinary experience, "we try to continue to
lead our lives as normally as possible".
His married daughter lives only a few miles away, his son remains at home,
though works full time, and his younger daughter has just returned to school.
"Each day is a celebration, in a sense, because they are days that I would not
have had."
GENE THERAPY FOR CANCER
TREATMENT
So, Why don’t we cure everybody?
FDA has not approved gene therapy because it has proven
dangerous.
Not a permanent cure
Our bodies can have an immune response to the vector and
DNA.
Many diseases are caused by multi-gene problems
Jesse Gelsinger
•Jesse suffered from a rare genetic
defect in which his liver could not clear
his body of ammonia
•In 1999 underwent gene therapy; was
inserted with a adenovirus carrying a
repair gene
•The virus attacked his organs
•4 days later, he died at the age of 18
•1st casualty of gene therapy
What is the Human Genome Project? Begun formally in 1990,
the U.S. Human Genome Project was a 13-year effort
coordinated by the U.S. Department of Energy and the
National Institutes of Health. The project originally was
planned to last 15 years, but rapid technological advances
accelerated the completion date to 2003. Project goals were
to:
•identify all the approx 20,000-25,000 genes in human DNA,
•determine the sequences of the 3 billion chemical base pairs,
•store this information in databases,
•improve tools for data analysis,
•transfer related technologies to the private sector, and
•address the ethical, legal, and social issues from HG Project
What's a genome? And why is it important?
A genome is all the DNA in an organism, including its genes.
Genes carry information for making all the proteins
required by all organisms. These proteins determine, among
other things, how the organism looks, how well its body
metabolizes food or fights infection, and sometimes even
how it behaves.
The particular order of As, Ts, Cs, and Gs is extremely
important. The order underlies all of life's diversity, even
dictating whether an organism is human or another species
such as yeast, rice, or fruit fly, all of which have their own
genomes and are themselves the focus of genome projects.
Because all organisms are related through similarities in
DNA sequences, insights gained from nonhuman genomes
often lead to new knowledge about human biology
http://www.ornl.gov/sci/techresources/Human_Genome/posters/chromos
ome/chooser.shtml