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DNA Technology
and Biotechnology
Biotechnology = refers to the
technology used to manipulate
DNA
 Procedures are often referred to as Genetic
Engineering
 DNA is the genetic material of ALL living things
 All organisms use the same genetic code
 Genes from one organism can be transcribed
and translated when put into another organism
Examples...
• Human genes (and other animals) are
routinely put into Bacteria in order to
synthesize products for medical
treatments and commercial use i.e.
human insulin, human growth hormone,
vaccines
“Recombined” DNA
 Recombinant DNA = refers to DNA
from two different sources being
combined
 Individuals receiving genes from other
species are called Transgenic
Vectors
• Vector = anything used to transfer a
gene into a host cell
Plasmids and Viruses are the most
common vectors used
What’s the Difference?
Virus: (Protein coat & DNA or RNA core) takes
over the host cell machinery by incorporating
itself into the host DNA to make more virus
particles
Plasmid: a small ring of DNA, incorporates
itself into the host DNA, cloned each time the
host DNA is reproduced
A vector must be capable of self replicating
inside a cell!
Tools of the Trade
• Restriction Enzymes: discovered in 1970’s in
bacteria
 Used naturally in bacteria as a defense
mechanism against viruses (bacteriophages)
 “Cut” DNA at specific base sequences
• Example: EcoR1 always cuts DNA at
GAATTC as shown:
Other restriction enzymes cut at different
sites, some examples are listed below:
•
•
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Enzyme
Bam HI
Hae III
Pst 1
Hind III
Cutting Sites
GGATCC
GGCC
CTGCAG
AAGCTT
Sticky Ends
• Sticky Ends & Recombinant DNA:
Fragments of DNA that have been cut
using restriction enzymes have unpaired
nucleotides at the ends called Sticky
Ends
 Sticky ends have complementary bases , so
they could rejoin
 If the vector and the gene to be cloned are both
cut with the same restriction enzyme, they will
both have complementary sticky ends
 After cutting, 2 DNA samples are mixed
 Result = Recombinant DNA
 The enzyme DNA ligase seals the fragments
together
Genomic Libraries
• Genomic Libraries: collection of all the
genes that an organism possesses
 Bacteria or viruses can be used to store
fragments of DNA from another species
PCR
• Polymerase Chain Reaction (PCR):
used to make numerous copies of small
pieces of DNA
• Requires = primers, DNA polymerase, &
nucleotides
How Does PCR Work?
 1. The DNA is heated to about 95O C to
separate the 2 strands of the double
helix
• 2. After the strands are separated, the
DNA is cooled to about 50OC and the
primers attach
• 3. Temp. is then raised to 70OC so the
polymerase will attach and copy the
strand
• 4. The DNA Replication process
repeats itself as the solution is heated
and cooled at regular intervals
DNA Fingerprints
• DNA Fingerprinting (RFLP Analysis):
banding pattern produced on a gel
representing the accumulation of DNA
fragments of various sizes
Gene Products and the Uses of
Genetic Engineering
 E. coli used to produce insulin
 Mammalian cells used to produce proteins such
as hormones
 Plant cells can be engineered with new
properties (insect resistance)
 Bovine Growth Hormone (BGH) used to
increase milk production in cows by about 10%
• Animal viruses can be engineered to carry a
gene for a pathogen’s surface protein so the
virus can be used as a vaccine
Gene Products and the Uses of
Genetic Engineering
 Techniques were used to map the human
genome through the Human Genome Project
 Could provide tools for diagnosis and possible
repair of genetic diseases
 Gene therapy could be used to cure genetic
diseases by replacing defective or missing
genes
Social and Ethical Issues
Of
Genetic Engineering
Harmful organisms may be accidentally
produced
Organisms that are released into the
environment may be engineered with genes
that will eventually kill them
There is currently little legislation on the use of
genetic screening and the use of information
produced by screening; could result in
discrimination
Social and Ethical Issues
Of
Genetic Engineering
This technology is increasing the ability to
diagnose genetic diseases pre-natally, adding
new complexity to the abortion controversy
Ethical questions have been raised over
whether we should modify the genes of
humans
Genetic screening and gene therapy are
expensive and may be unavailable to the poor
Social and Ethical Issues
Of
Genetic Engineering
Biological weapons could be created using
biotechnology
The debate over stem cells would fall into this
category as well as cloning – limited legislation
already exists
DNA Fingerprinting
 Facts:
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There are over 10 trillion cells in your body
Every cell has the same DNA inside
There are over 3 billion base pairs in your DNA
Your DNA is spread over 46 chromosomes
Your DNA is unique to you! (Unless you have
an identical twin)
How to make a DNA fingerprint:
 1. Pour restriction enzymes into DNA
 2. Separate DNA fragments using an
agarose gel (small pieces will move farther)
using an electrical current
 3. Add probes for certain DNA Markers
 4. Create an x-ray film of your results
called an Autoradiograph
What do you end up with?
 A film with bands on it that represent the
different fragment lengths of DNA.
 The bands show where the probes attached
themselves to the DNA.
What are DNA Fingerprints Used
For?
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Criminal Cases (Murder, Rape, Etc.)
Paternity cases (Who's yo daddy?)
Free wrongly incarcerated inmates
Create DNA Profiles
Construct Genetic Databases (Convicted
felons)
History
 1996 Dr. Ian Wilmut - first to successfully
clone an adult mammal
 “Dolly” the sheep: born 1996, died
(euthanized) Feb. 14, 2003
 Named after Dolly Parton (donor cell was
taken from an adult mammary gland)
How the Process Works
 1. Take a donor cell from an adult.
 2. Starve the cell of nutrients and it will
become dormant.
 3. Remove an unfertilized egg cell from a
female.
 4. Replace the nucleus of the unfertilized
egg cell with the nucleus of the adult donor
cell.
How the Process Works
 5. “Trick” the egg cell into thinking that it
was fertilized naturally (electric shock
works well).
 6. The egg will now grow and divide
(mitosis) to form an embryo.
 7. Implant the dividing egg cell into a
surrogate mother.
How the Process Works
 8. Wait out the normal gestation period.
 9. At the completion of the pregnancy, the
organism that is born will have the same
DNA as the original donor.
Problems So Far / What we’ve
Learned
 Dolly only lived 7 years, less than 1/2 the
normal life expectancy
 Dolly developed early onset arthritis
 Successful cloning has a high failure rate
 Telomere = found at the tips of
chromosomes
Significance of the Telomere
 It gets shorter every time the DNA
replicates before Mitosis
 Acts as a “Biological Clock”
 When the telomere is gone the cell no
longer divides
 An enzyme called telomerase is responsible
for the eating away of the telomere
Possible Medical Applications
 Organ and tissue transplants
 Taking a specialized cell and turning it into
any tissue or organ you want
 Genetically resistant foods (Bananas)
Stem Cells
 What are human embryonic stem cells?
Stem cells are cells that have the remarkable
potential to develop into many different cell
types in the body.
 The two categories of stem cells include
Embryonic and Adult Stem Cells and
possibly a third, cord-blood-derived
embryonic-like stem cells (CBEs).
Stem Cell Treatment
 Medical researchers believe that stem cell
research has the potential to change the face
of human disease.
 Medical researchers anticipate being able to
use technologies derived from stem cell
research to treat Cancer, Parkinson’s
Disease, Spinal Cord Injuries, and muscle
damage.
The Controversy
 There exists a widespread controversy over
stem cell research that emanates from the
techniques used in the creation and usage of
stem cells. Embryonic Stem Cell research
is particularly controversial because, with
the present state of technology, starting a
Stem cell line requires the destruction of a
human embryo and/or therapeutic cloning.