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Biotechnology
Biotechnology
• “Biotechnology means any
technological application that uses
biological systems, living
organisms, or derivatives thereof,
to make or modify products or
processes for specific use.”
Bacteria with Plasmid
1 = bacteria regular chromosome
2 = plasmids
Plasmid
• Small circular DNA separate
from the larger bacterial
chromosome
Vector
A Vector = an
agent that can
carry
something
from one
organism to
another.
Why is a plasmid important in
biotechnology?
• It may be used
as a vector, to
carry other
genes to another
cell.
Gene therapy is being used in
many ways. For example, to:
• Replace missing or defective genes;
• Deliver genes that speed the destruction of cancer cells;
• Supply genes that cause cancer cells to revert back to
normal cells;
• Deliver bacterial or viral genes as a form of vaccination;
• Provide genes that promote or impede the growth of new
tissue; and;
• Deliver genes that stimulate the healing of damaged
tissue.
Transfer of DNA
R plasmids
• This type of plasmids carry genes for
enzymes that destroy antibiotics such
as penicillin and tetracycline.
• So…Bacteria containing R
plasmids become resistant to
many antibiotics.
Steps for Gene Therapy
• 1. Isolate
plasmid
2. DNA is isolated
from plant,
animal, or other
bacteria
Animation
• LINK: Isolating
Plasmid DNA from
Bacteria
3. Gene is
inserted into
plasmid
RECOMBINANT
DNA = plasmid +
inserted DNA
4. Recombinant DNA plasmid is put
into bacterial cell
• DNA cloning animation
• 5. Cell multiplies with
recombinant DNA
Restriction enzyme
• (or restriction endonuclease) is an enzyme
that cuts double-stranded DNA at a specific
location
Restriction Enzymes
Different Restriction Enzymes
Several
hundred
different
kinds
LINK:
RECOGNITION
SITES OF
RECTRICTION
ENZYMES
Sticky Ends
“Sticky Ends”
Complementary
bases join
plasmid an new
DNA to form
recombinant
DNA
Restriction site
= place where
the two
fragments join
Ligase Enzyme
Joins the
sticky ends
Recombinant DNA
• Plasmid DNA + other
organism DNA
• LINK: rDNA
DO LAB ACTIVITY
Chocolate Flavored Cherries: An
exercise in Recombinant DNA
Technology
Read intro
and do PreActivity First
Gel Electrophoresis
• Electrophoresis is a technique used to
separate the DNA fragments according to
their size and
charge.
Gel Electrophoresis
• Gel Electrophoresis Virtual Lab
• Gel Electrophoresis
Pour Agarose Gel, cool to set
Gel comb is used to make holes in
the gel
load the wells with DNA samples
Ready to Run
• DNA flows
from – to +
end of the gel
• Cutting our DNA with restriction enzymes
makes a unique set of restriction
fragments. The number of restriction
fragments and their sizes reflect the
specific sequence of nucleotides in your
DNA.
• The
smaller
the
molecules
the faster
they move
DNA Fingerprinting
Each person produces a specific pattern of
bands of DNA sequence lengths
Examine the results
• UV Transilluminator, and the UV light
shines up through the gel.
Click and Drag to find the suspect
Murder at Rodman Dam
•http://www.dnalc.or
g/ddnalc/resources/
shockwave/dnadete
ctive.html
Do any columns match?


7 suspects
Crime scene
bloodstain is in the
middle
Which suspect is guilty?
Which is the father?




M=mother, Fern
C=child
F1=Ross
F2=Rick
• Some Examples of DNA Uses for
Forensic Identification
• Identify potential suspects whose DNA
may match evidence left at crime scenes
• Exonerate persons wrongly accused of
crimes
• Identify crime and catastrophe victims
• Establish paternity and other family
relationships
More Uses
• Detect bacteria (like Salmonella) and other
organisms that may pollute air, water, soil,
and food
• Match organ donors with recipients in
transplant programs
• Determine pedigree for seed or livestock
breeds
More Uses
• Authenticate consumables such as caviar
and wine
• Detect disorders like cancer, cystic fibrosis
• Identify endangered and protected species
as an aid to wildlife officials (could be used
for prosecuting poachers)
Repeat Fragment Length
Polymorphism
• Making a RFLP:
• blood, hair root, or
other biological
sample.
• cut with a restriction
enzyme
• Separate with gel
electrophoresis
OTHER FRAGMENTS: VNTR’s
• Variable number of tandem
repeats (VNTRs) can be used in a
gel electrophoresis to show
different patterns
Southern Blot Animation
• LINK: Animation Quiz 5 - Southern Blot
Paternity Testing: Call Montel
• NICE LINK Paternity
Testing
Would any one else have the same
pattern as you?
• No…unless you are an
identical twin.
DNA PROBES
• a single-stranded
DNA molecule (like
ATTGGAC) used in
laboratory experiments
to detect the presence
of a specific sequence
among a mixture of
other singled-stranded
DNA molecules.
USES of DNA PROBES
• Detect infectious diseases
• Detect genetic diseases
• HLA typing (suitable donor for
stem cell blood typing)
• Paternity (find the father)
• Cancer detection
fluorescent probe
DNA
probe
PCR
• Polymerase Chain
Reaction• take a small sample of
DNA an amplify it take
make a larger amount
• Sample does not have to
be pure and can be
partially degraded.
Start with very small amount
Doubles each cycle
PCR Inventor
Kary Mullis
Nobel Prize
1998
PCR Animations
• LINK: Animation Quiz 6 - Polymerase
Chain Reaction
• LINK: Polymerase Chain Reaction
Denaturation: high
temp. breaks apart
DNA strands
Annealing: cool to
add primers
Extensions: Taq
DNA polymerase
synthesizes new
DNA strands
Taq Polymerase
• An enzyme used in PCR when it is heated
• Thermus aquaticus (hence the
abbreviation "Taq"), a bacterium that lives
in hot springs and hydrothermal vents, Taq
was identified as the first polymerase able
to withstand the denaturing conditions
required during PCR.
12.13 HUMAN GENOME
• STARTED 1990
• Completed in 2003, the Human
Genome Project (HGP) was a 13year project
• Finished two years ahead of
schedule
HGP Goals
• identify all the
approximately 35,000
genes in human DNA
“Junk” DNA
• It is thought 97% of the
DNA is noncoding DNA
• Promotors, enhancers,
junk DNA, introns
Telomeres
• Regions of
highly
repetitive DNA
at the end of a
linear
chromosome
for protection
Telomeres
• The telomeres of
humans consist
of as many as
2000 repeats of
the sequence
5' TTAGGG 3'.
“Jumping Genes”
• Also called Transposons
are sequences of DNA that
can move around to
different positions within the
genome of a single cell,
• The reddish streaks on these
corn grains are caused by
transposons.
Barbara McClintock
• The Nobel Prize in Physiology or Medicine
1983 for finding jumping genes
HGP Human Genome Project
HGP Purposes
• 1- determine the
sequences of the
3 billion chemical
base pairs that
make up human
DNA
HGP Goals
•2. store this
information in
databases (sex
offenders, soldiers)
HGP Goals
•3 - transfer related
technologies to the
private sector (see
next slide)
Products from Recombinant DNA
• 1. Human Insulin Recombinant
DNA: Example Using Insulin
• 2. Human Growth Hormone
• 3. Interleukin-2 (chemical made
by T cells) to treat cancer
• 4. Factor VIII to treat hemophilia
• 5. Vaccines
GM Organisms
• Genetically Modified
= has acquired one
or more genes by
artificial means
• EXAMPLE: Golden Rice
has vitamin A
(β-carotene) in the
grain
A tobacco plant
which has been
genetically
engineered to
express a gene
taken from
fireflies.
GloFish
GloFish: the
first
genetically
modified
animal to be
sold as a
pet.
Flavr Savr Tomato
• Contains the
gene which
inhibits the
fruit to soften,
therefore, the
fruit stays firm
longer.
Bt Corn
• GOOD: protection
from pests, tolerance
to pesticides, or
improve its quality.
• BAD: suggested that
pollen from Bt corn
may have toxic effects
on larvae of the
monarch butterfly.
Ti Plasmid
• LINK The Ti Plasmid
•
USE: Ti plasmid is a circular
plasmid that uses
Agrobacterium tumefaciens to
transduce its genetic material
to plants
Decaffeinated Coffee Plants
Three enzymes
are involved in
caffeine
biosynthesis in
coffee plants —
feasible to produce coffee
beans that are intrinsically
deficient in caffeine.
Leaf Disk Method for Agrobacterium-Mediated Transformation of Higher Plant Cells
Leaf Disk Preparation
Regeneration of Shoots
Co-cultivation with Agrobacterium
Selection for Transformation
Adaptation to growth in the Greenhouse
Photographs by Dr. Paul Bottino
Plant Tissue Culture
• The Ti Plasmid animation
• Ti = “tumor inducing”
Plant gall
Bt Crops
What is Bt?
Bacillus thuringiensis (Bt) is a common soil
bacterium that produces crystals containing
proteins that are toxic to certain insects.
Bt corn, and Bt potatoes are being grown in
the United States, Canada, Argentina, South
Africa, France, and Spain.
Transgenic Organisms
Transgenic Organisms
• A transgenic
organism
carries in all its
cells a foreign
gene that was
inserted by
laboratory
techniques.
GM Rabbit
GFP stands
for green
fluorescent
protein. Her
name is Alba.
Gene Therapy
• Alteration of an individual’s genes
• Who will have access to it? Only
the rich?
• Can we use it to enhance a
physical ability (superman)?
How might GM organisms harm
human health or the environment?
• 1. allergies from inserted genes
• 2. pass genes to closely related
species in wild areas
• 3. may pass mutations to humans
who eat the food
• 4. make the soil toxic.
HGP Goals
• address the ethical, legal,
and social issues (ELSI)
that may arise from the
project.
Other Ethical Questions DNA
technology might raise:
• Who should have access to personal
genetic information, and how will it be
used?
• Who owns and controls genetic
information?
• How does personal genetic information
affect an individual and society's
perceptions of that individual?
• Should testing be performed when no
treatment is available?
• Do people's genes make them behave in a
particular way?
• Who owns genes and other pieces of
DNA?
ANIMATION
• LINK: Animation Quiz 1 - Cloning a Gene
CLONING ANIMALS AND
HUMANS
3 Types of Cloning
• The following three types of cloning
technologies will be discussed:
• (1) recombinant DNA technology or DNA
cloning,
• (2) reproductive cloning, and
• (3) therapeutic cloning.
Reproductive cloning
• uses "somatic cell nuclear transfer"
(SCNT) to create animals that are
genetically identical
• transfer of a nucleus from a donor adult
cell (somatic cell) to an egg which has no
nucleus.
• If the egg begins to divide normally it is
transferred into the uterus of the surrogate
mother.
Extracting nucleus
• it took 276 attempts
before the
experiment was
successful
Dolly – died at age 6
Dolly, the first mammal to
be cloned from adult DNA,
was put down by lethal
injection Feb. 14, 2003.
suffering from lung cancer
and crippling arthritis.
most Finn Dorset sheep
live to be 11 to 12 years of
age,
Cloning Process
• Cloning Animation
Severino Antinori
• Italian fertility specialist
Severino Antinori 2001.
• The Italian medical authorities
warned that Dr Antinori risked
losing his right to practice in
Italy because of his plans to
clone human beings.
Therapeutic
Cloning
Therapeutic cloning
• does not strive to make whole humans.
• Instead, it makes embryos as a source of
embryonic stem cells for therapeutic purposes.
Because embryonic stem cells can grow into any
body cell, they might be cultured into nerve cells,
skin cells, even hair follicles for the bald. The
obvious use of therapeutic cloning would be
treating deadly diseases like diabetes and
Parkinson's, where a specific type of cell has
died.
Stem Cells