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GENE TECHNOLOGY
GENETIC ENGINEERING

Human manipulation of the DNA code of an
organism in order to:
 Make
transgenic organisms (done using
recombinant DNA)
 Clone
an organism
 Perform
Gene therapy
TRANSGENIC ORGANISMS

Organisms which express a gene from another
organism

Insert gene of interest into another organism,
receiving organism now makes the protein from
that gene
TRANSGENIC ORGANISMS

Plants with “resistance” genes or “insecticide
genes”
EXAMPLE:
DNA alteration is done in
plants to develop seeds
which are resistant to
herbicides the farmer
sprays to destroy weeds.
TRANSGENIC ORGANISMS

Cows with extra copies of growth hormones
TRANSGENIC ORGANISMS

Insulin
making
bacteria
AND….MOST IMPORTANTLY????

Cool glow in the dark mice (or worms)!
MAKING INSULIN FOR DIABETICS
Diabetes: mutated insulin gene which creates a
dysfunctional insulin protein;
 RESULT: no or low amounts of insulin protein
made


QUESTION: How can we get insulin that is of
“consistent strength”, high purity and easy to
make in large quantities (low cost).
USE BACTERIA AS AN “INSULIN FACTORY”
Bacteria have circular pieces of DNA called
Plasmids
 They can replicate, transcribe and translate any
genes on the plasmid

WHAT DOES A PLASMID LOOK LIKE?
HOW DO WE WORK WITH PLASMIDS?

We can
actually take
out the
plasmid, add
genes and
reinsert the
plasmid into
the bacteria
RESTRICTION SITES

In plasmids there are specific sequences called
restriction sites. Restriction sites are locations
on a DNA molecule containing specific
sequences of nucleotides
restriction site
RESTRICTION ENZYMES

Restriction enzymes recognize the sites and cut
the DNA at that site
RESTRICTION ENZYMES ARE SPECIFIC

Each restriction enzyme recognizes and cuts a
different sequence
Examples:
Restriction Enzyme
Restriction Site
EcoRI
GAATTC
Hind III
AAGCTT
BamH1
GGATCC
HOW RESTRICTION ENZYMES WORK

Restriction
enzymes
recognize
the sites and
cut one
strand of the
DNA at that
site
G AATTCGACTAGCGAT
GTGGATCGATCTTAA GCTGATCGCTA
CACCTAGCTA
HOW MANY PIECES DO YOU GET?
G
GAATTC
AATTC
GACTAGCGAT
GACTAGCGAT
GTGGATCGATCTTAAGCTGATCGCTA
GTGGATCGATCTTAA
GCTGATCGCTA
CACCTAGCTA CACCTAGCTA
SINGLE STRANDED ENDS ARE “STICKY”
–Want to bind to complementary bases
G
GTGGATCGATCTTAA
CACCTAGCTA
AATTCGACTAGCGAT
GCTGATCGCTA
ADDING THE INSULIN GENE

We can take advantage of this and insert any
gene we want into the breaks
Insulin
SEALING THE ENDS OF THE INSULIN GENE

What enzyme can we use to “seal the gaps”
between plasmid DNA and insulin DNA?
Insulin
Ligase
TRANSFORMATION

Put plasmid back into bacteria (a process
called transformation)

Bacteria will transcribe and translate our
insulin gene even though the insulin protein
doesn’t do anything for a bacterial cell.

Then we can take out the insulin protein and
use it to treat diabetics.
MAKING INSULIN USING A VECTOR
 Step
1. ____Isolate_________ the plasmid DNA
and human (or other ) gene of interest
 Step 2 Use ___restriction___ enzymes to cut both
the __plasmid__ and the gene DNA you wish to
insert
 Step 3. Seal the sticky ends using ____DNA
Ligase___________
 The recombinant DNA plasmids are then reinserted
and reincorporated into the bacteria.
RECOMBINANT DNA SUMMARY

Recombinant
DNA (rDNA) is
DNA that has
been created
artificially. DNA
from two or more
sources is
incorporated into
a single
recombinant
molecule.
RECOMBINANT DNA – WHY???
Better Crops (drought & insect resistance)
 Larger livestock (cows, chickens, hogs)
 Recombinant Vaccines (ie. Hepatitis B)
 Prevention and cure of sickle cell anemia
 Prevention and cure of cystic fibrosis
 Production of clotting factors
 Production of insulin
 Plants that produce their own insecticides
 Germ line and somatic gene therapy

WHAT IS A CLONE?

An organism that is
genetically identical to
its parent
HOW DOES CLONING WORK?

Mammals usually fuse info from two parents
(sexual reproduction)

Cloning takes all the chromosomes from 1
parent, (but does use “different cells”)
HOW DO WE CLONE?
Sheep 1
Take 1 body cell
(udder)
Extract Nucleus
Sheep 2
Take 1 egg cell
Remove nucleus
HOW DO WE CLONE?
Zap to stimulate
cell division
Inject nucleus
into Egg
Implant embryo into
surrogate sheep
(sheep 3)
NOW WE WAIT FOR DOLLY….
Which sheep is Dolly identical to??
Why?
Which sheep have to be female?
CLONING DOLLY (SUMMARY)
Somatic cell (2N chromosomes) was used from
one animal (this is who we are cloning).
 We extract and keep the nucleus (get rid of the
rest)
 Egg cell from a second animal (nucleus
contains only 1n chromosomes)
 Extract and get rid of nucleus and keep the cell
 Fuse the two together  zygote  put into a
3rd sheep to gestate.

ORIGINAL, CLONE AND
SNUPPY
SURROGATE
SURROGATE
WHY DO WE CLONE?

Scientific
experimentation

Maintain a
genetic line (good
sire or dam)
Two mini clone pigs,
nine days after they
were born. Their
internal organs are
quite similar with
those of human
beings and are used
for organ transplant
experiments.
CLONING

Coming next?????
WHAT DO WE KNOW ABOUT THE HUMAN
GENOME>
HUMAN GENOME PROJECT

What it did do: Tell us each an every nucleotide
of the human genome (all 3.2 billion)
 GOALS







WERE:
identify all the approximately 20,000-25,000 genes in human DNA,
determine the sequences of the 3 billion chemical base pairs that make up human DNA,
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 (ELSI) that may arise from the project.
What it did not do: Tell us what it all means!!!
HUMAN GENOME PROJECT…WHERE ARE WE?
Now we have to break it down and determine:
- which pieces are genes
- which pieces are junk
- what info the genes hold.
WHERE DO WE STORE ALL THIS INFORMATION?

Bioinformatics
BIOINFORMATICS
The application of computer
science to the field of
molecular biology.
Bioinformatics now includes
the creation and
advancement of databases,
algorithms, computational
and statistical techniques,
and theory to solve formal
and practical problems
arising from the
management and analysis of
biological data.
BLAST SEQUENCE

BLAST finds regions of similarity between
biological sequences.

You can use a sequence of nucleotides (nucleotide
BLAST) or amino acids (protein BLAST)

For example, following the discovery of a
previously unknown gene in the mouse, a scientist
will typically perform a BLAST search of the human
genome to see if humans carry a similar gene;.
WHAT ELSE CAN WE DO WITH “DNA PIECES”

Each of you has similar genes, but within the
genes are unique sequences that identify “you”

You receive some sequences from your mother,
and some from your father.

In most offspring, some can be seen from each
parent. We can establish paternity, guilt, etc.
DNA FINGERPRINTING
Used to compare two people’s DNA
 Used in paternity cases
 Used for crime scene analysis

DNA FINGERPRINT
IDEA BEHIND FINGERPRINTING

Based on the idea that EVERYONE’s DNA is
unique, like a fingerprint

BUT related individuals will have more
similarities

HOW IS A FINGERPRINT DONE?
CREATING A FINGERPRINT
Get a sample of DNA and digest it with
restriction enzymes
 If everyone’s DNA is unique, the enzyme will cut
each persons DNA differently
 Example:


TCATGAATTCATTGCCGAATTCCGTGAATCCAGAATTCGGACTA

TCATGAAGTCATTGCCGAATTCCGTGAATCCAGACTTCGGACTA
RESULT: DIFFERENT FRAGMENTS OF DNA

Run cut up DNA on through electrophoresis

Click here for animation

Small pieces travel fast and move further down
the gel slab.

Large pieces move slower and stay closer to the
injection point.
DNA LADDER

A DNA ladder is a
solution of DNA
molecules of different
lengths used in agarose
gel electrophoresis. It is
applied to an agarose
gel as a reference to
estimate the size of
unknown DNA
molecules.
VNTR SEQUENCES

Although 99.9% of human DNA sequences are
the same in every person, enough of the DNA is
different to distinguish one individual from
another, unless they are monozygotic twins.[2]
DNA PROFILING (FINGERPRINTING) AND VNTR
LOCATIONS

DNA profiling uses repetitive ("repeat")
sequences that are highly variable,[2] called
variable number tandem repeats (VNTRs),
VNTR locations on a gene (loci) are very similar
between closely related humans, but so
variable that unrelated individuals are
extremely unlikely to have the same VNTRs.
DNA FINGERPRINT



While DNA in all humans is similar there are differences
DNA fingerprinting can be used to identify a child’s
parents.
In this example (next page) , a family consists of a mom
and dad, two daughters and two sons. The parents have
one daughter and one son together, one daughter is
from the mother’s previous marriage, and one son is
adopted, sharing no genetic material with either parent.
After amplifying the VNTR DNA from each member of the
family, it is cut with a restriction enzyme and run on an
agarose gel. Here are the results:
DNA FINGERPRINT

Which child is adopted? Who are
the children of this Mom and Dad
WHY USE DNA FINGERPRINTS?
Anthropologists can trace human origins and
migrations
 Environmental conservationists track
migratory patterns and movements of
endangered species
 Paternity Testing
 Forensics testing

USING DNA LADDERS TO FIND
GENE MUTATIONS: PROBES

To find a target gene mutation (like a
gene we think causes a disease) in a
sample of DNA, scientists use a DNA
probe - a length of single-stranded
DNA that matches part of the gene
and is linked to a radioactive atom.
The single-stranded probe seeks and
binds to the gene. Radioactive signals
from the probe are then made visible
on x-ray film, showing where the probe
and gene matched.
GENE THERAPY
Taking genetic testing one step further
 Gene therapy tries to FIX the genetic problem

HOW DO WE “FIX” A GENE?
Take a virus
that naturally
infects the type
of cells that are
defective.
Remove all the
virus’s DNA.
Replace it with
correct copy of
defective gene
Infect “disease
cells with the
“correct gene”
APPLICATIONS
1.
2.
3.
Cystic fibrosis
Hemophilia
Cancer
CYSTIC FIBROSIS

Used a virus to carry normal CFTR gene to
affected lung cells

Only temporary
HEMOPHILIA
Good candidate since the the disease is caused
by a single known genetic defect.
Has been successful in dogs, but not humans

Latest approach is using nanotechnology
(nanoparticles containing the gene coupled
with a genetic element that helps with insertion
of the gene (no longer use a virus).
CANCER

Still experimental

Researchers are trying to improve the body's
natural ability to fight the disease or to make
the cancer cells more sensitive to other kinds
of therapy (chemotherapy or radiation).
WANT TO KNOW MORE?

http://www.ornl.gov/sci/techresources/Human
_Genome/medicine/genetherapy.shtml
APPLICATIONS OF GENETIC ENGINEERING

Predominantly found in

A. Agriculture (transgenics and cloning)
DISEASE RESISTANT PLANTS
 PLANTS WITH HIGHER YIELD (MORE FOOD PER ACRE)
 PEST RESISTANT PLANTS (CONTAIN A GENE WITH AN INSECTICIDE).
 CLONING high producing animals (high milk, meat, etc)


B. Medicine (gene therapy, cloning, transgenics)
Gene Therapy
 Cloning animals for organ transplants
 Cloning
 Production of the oncomouse (a mouse used to study cancer)

ETHICS OF GENE TECHNOLOGY

Are we blurring the lines between species by creating transgenic
combinations?

What are the known health risks associated with transgenics?

What are the long-term effects on the environment when transgenics
are released in the field?

Are we inflicting pain and suffering on animals when we create
certain types of chimeras (multiple species animals)?

Will transgenic interventions in humans create physical or behavioral
traits that may or may not be readily distinguished from what is
usually perceived to be “human”?