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NOTES - CH 15 (and 14.3):
DNA Technology (“Biotech”)
BIOTECHNOLOGY: the use
of living organisms or their
components to do practical
tasks
“TRADITIONAL” BIOTECH:
-microorganisms to make
wine / cheese
-selective breeding of
livestock
-production of antibiotics
**Practical goal of
biotech =
improvement of
human health and
food production
DNA Technologies:
1) Making a recombinant
DNA molecule;
2) Gene therapy;
3) DNA fingerprinting;
4) Cloning.
Recombinant DNA:
• Combining fragments of
DNA from different
sources;
• Result: organisms with
their DNA + foreign
DNA…such organisms are
known as: TRANSGENIC
ORGANISMS.
Example of transgenic organism:
 Tobacco plant
that contains a
gene from a
firefly – it glows!
BIOLUMINESCENT CAT!
“Toolkit” for recombinant DNA technology
involves:
-restriction enzymes
-DNA vectors
-host organisms
RESTRICTION ENZYMES = enzymes that
recognize and cut short, specific DNA
sequences
Restriction Enzymes…
are used to cut out a specific DNA
fragment from an organism’s genome;
 recognize sequences that are
“palindromic” (the same letters
backward and forward);
 typically cut sequences in a “staggered”
manner so that the two ends of the
fragments are single-stranded;
Restriction Enzymes (cont.)…
 this creates “sticky ends” so that the
DNA fragment from one organism will
be complementary to the DNA fragment
from another organism.
(complementary base pairing)
Gene Splicing:
• GENE SPLICING = rejoining of DNA
fragments after cutting with restriction
enzymes – foreign DNA is recombined into
a bacterial plasmid or viral DNA
VECTORS = carriers for moving DNA
from test tubes back into cells
-bacterial plasmids (small, circular
DNA molecules that replicate within
bacterial cells)
-viruses
HOST ORGANISMS:
bacteria are commonly
used as hosts in genetic
engineering because:
 bacterial cells are simple, and grow
quickly, replicating and expressing any
foreign genes they carry.
Gene Cloning:
• Once the foreign
DNA has been
transferred into the
host bacterial cell, it
replicates every time
the cell divides;
• CLONES =
genetically identical
copies of a gene
Gene Expression:
• In addition to copying the introduced foreign
gene, bacterial cells will also EXPRESS the
genes (make the protein the gene encodes!)
• EXAMPLE: if the gene for human insulin is
inserted into a bacterial
plasmid and then into a
host bacterial cell, that cell
will start to make
HUMAN INSULIN!
Steps Involved in
Cloning a Human Gene:
1) Isolate human gene to clone;
plasmid
2) Isolate plasmid from bacterial cell;
Human gene
3) Add a restriction enzyme to cut out human
gene & add same R.E. to open up bacterial
plasmid (creates complementary “sticky ends”);
4) Combine human gene with bacterial
plasmid;
Cloning a Human Gene (cont.)…
5) Insert recombinant DNA plasmid back into
bacterial cell;
6) As bacterial cell reproduces, it makes copies
of the desired gene…and expresses that
gene (makes whatever protein the gene
encodes)!
Applications of DNA
Technology:
•
•
•
•
•
Recombinant bacteria in industry;
Recombinant bacteria in medicine;
Recombinant bacteria in agriculture;
Transgenic animals;
Transgenic plants.
Recombinant bacteria in
industry:
• Bacteria that can:
 break down pollutants;
 degrade oil spills;
 extract minerals from ores.
Recombinant bacteria in
medicine:
• Bacteria that have received human genes
and produce:
 human growth hormone;
 insulin to treat
diabetes;
 the amino acid
phenylalanine.
Recombinant bacteria in
agriculture:
• Bacteria that:
 protect crops against frost;
 produce natural fertilizers;
 prevent crops from spoiling after
harvest.
Transgenic animals:
• Engineer / produce animals with
human diseases so that they can be
studied in detail.
Transgenic plants:
• Plants that are engineered to:
 resist herbicides;
 produce internal pesticides;
 increase protein production.
Other DNA Technologies:
•
•
•
•
•
Polymerase Chain Reaction (PCR);
Human Genome Project;
Gel Electrophoresis;
Gene Therapy;
DNA Fingerprinting
The Polymerase Chain Reaction
(PCR)
 allows any piece of DNA to be
quickly copied many times in
the lab;
PCR (continued)…
 BILLIONS of copies of DNA are produced
in just a few hours (enough to use for testing);
In 6 cycles of PCR:
cycle 1: 2 copies
cycle 2: 4 copies
cycle 3: 8 copies
cycle 4: 16 copies
cycle 5: 32 copies
cycle 6: 64 copies
cycle 20: 1,048,576!!
Polymerase Chain Reaction (PCR)
PCR is highly specific;
only a small sequence
is amplified
 only tiny amounts of
DNA are needed.
Starting materials for PCR:
• DNA to be copied
• Nucleotides (A,G,C,T)
• Primers
• DNA polymerase
Applications of PCR:
 analyze DNA from tiny amounts of
tissue or semen found at crime scene;
 analyze DNA from single embryonic cells for
prenatal diagnosis;
 analyze DNA or viral genes from cells infected
with difficult to detect viruses such as HIV;
 used extensively in Human Genome Project
(14.3)
PCR works
like a
copying
machine for
DNA!
Analysis of Cloned DNA:
Gel electrophoresis
 separates DNA molecules based on
SIZE
 a mixture of DNA fragments will be
sorted into bands, each consisting of
DNA molecules of the same length
YOUR DNA
MY DNA
Steps Involved in DNA Fingerprinting:
1) Collect DNA from a sample;
2) Perform PCR if necessary to
make more DNA;
3) Cut DNA apart using RE’s
**Junk DNA (introns) will be cut at different
places for different people, therefore
producing different size fragments
DNA Fingerprinting (cont.)…
4) Electrophoresis is used to separate DNA
pieces on a gel to create a banding pattern;
5) Photo of DNA gel is taken as evidence;
6) Banding patterns can then be compared.
Sample Sample
1
2
DNA_DetectivePC.exe
Gene Therapy:
• GENE THERAPY = the insertion of normal
genes into human cells to correct genetic
disorders
• Diseases treated include:
 cystic fibrosis
 SCID
(immune deficiency)
Biotech Today &
Tomorrow
• Experimental
• Ethical issues
• Research funding
• Who can afford
treatment?