E - Bio @ Horton
Unit – Molecular Genetics
Notes – DNA Technology
AP Biology
DNA Technologies
A. Recombinant DNA Technology
1. Recombinant DNA (rDNA) contains
DNA from two different sources.
2. To make r DNA, technician selects a
vector.
3. A vector is a plasmid or virus used to
transfer foreign genetic material into a
cell.
4. A plasmid is a small accessory ring of
DNA in the cytoplasm of bacteria.
5. Plasmids were discovered in research
on reproduction of intestinal bacteria
Escherichia coli.
6. Introduction of foreign DNA into vector
DNA to produce rDNA requires two
enzymes.
a. First restriction enzyme is a
bacterial enzyme that stops
viral reproduction by cleaving
viral DNA.
1. Restriction enzyme is
used to cut DNA at
specific points during
production of rDNA. (See
in-class restriction
enzyme activity)
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2. It is called restriction enzyme because it restricts growth of viruses.
b. DNA ligase enzyme seals DNA fragments into the gap created by the restriction
enzyme.
7. Restriction enzymes cleave vector (plasmid) and foreign (human) DNA.
a. Cleaving DNA makes DNA fragments ending in short single-stranded segments
(sticky ends).
b. Sticky ends allow insertion of foreign DNA into vector DNA.
8. The foreign gene is sealed into the vector DNA by DNA ligase.
a. Treated cells take up plasmids, and then bacteria and plasmids reproduce.
b. Eventually, there are many copies of the plasmid and many copies of the foreign
gene.
c. When DNA splicing is complete, an rDNA (recombinant DNA) molecule is
formed.
B. Plasmid Vector Compared to Viral Vector
1. A clone is a large number of molecules, cells, or organisms that are identical to the
original.
2. Bacterial cells take up recombined plasmids when treated with CaCl2 to make them more
permeable.
3. If the inserted gene is replicated and expressed, we can recover the cloned gene or
protein product.
4. Viruses can be a vector to carry rDNA into bacterial cells.
a. Bacteriophages are bacteria that infect bacteria.
b. When a virus invades a cell, the viral DNA is released and enters the cytoplasm.
c. Inside cytoplasm of the host cell, viral DNA may direct reproduction of more
viruses.
d. Each virus in the bacteriophage clone contains a copy of the cloned gene.
C. The Polymerase Chain Reaction
1. The polymerase chain reaction
(PCR) uses the enzyme DNA
polymerase to carry out
multiple replications (a chain
reaction) of target DNA.)
2. PCR can create millions of
copies of a single gene or a
specific piece of DNA in a test
tube.
3. PCR is very specific -- the
targeted DNA sequence can
be less that one part in a
million of the total DNA
sample; therefore a single
gene can be amplified using
PCR.
4. The PCR does not replace
gene cloning; cloning produces
many more copies of a gene
and is used whenever a large
quantity of a gene or a protein
product is needed.
5. Before carrying out PCR,
primers must be available.
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a. Primers are sequences of 20 bases complementary to bases on either side of
"target DNA."
b. Primers are needed because DNA polymerase only continues ore extends the
replication process.
c. After primers bind to the DNA strand, DNA polymerase copies the target DNA.
6. Most labs have PCR machines; automation requires temperature-insensitive DNA
polymerase an enzyme that can stand high temperatures, to separate double-stranded
DNA.
D. Analyzing DNA (Also refer to DNA Fingerprinting Lab Activity)
1. An entire genome of an individual can be subjected to DNA fingerprinting.
2. DNA fingerprinting is the technique of using DNA fragments lengths, resulting from
restriction enzyme cleavage, to identify particular individuals.
a. DNA is treated with restriction enzymes to cut it into fragments.
b. Each organism's DNA results in different-sized restriction fragment length
polymorphisms (RFLP's).
c. During gel electrophoresis, fragments separate according to length, resulting in a
pattern of bands.
d. Radioactive probes allow specific sequences to be separated from all other
fragments, and the resulting pattern to be recorded on X-ray film
(autoradiography).
e. DNA fingerprinting can identify deceased individuals from skeletal remains,
perpetrators of crimes from blood or semen samples, and genetic makeup of
long-dead individuals or extinct organisms.
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3. Following a PCR, DNA segments can be analyzed.
4. Therefore, PCR amplification and
analysis is used to
a. detect viral infections,
genetic disorders, and
cancer, and in forensic
laboratories;
b. estimate evolutionary
relationships of present-day
species by comparing
nucleotide sequences;
c. determine the nucleotide
sequence of human genes: the Human Genome Project; and
d. conduct molecular paleontology by determining the evolutionary history of human
populations.
Products of DNA Technologies
A. Transgenic Plants
1. Genetically engineered organisms can produce biotechnology products.
2. Free-living organisms that have had a foreign gene inserted into them are transgenic.
B. Transgenic Bacteria
1. Bacteria are grown in large vats.
a. When foreign genes are inserted, much product can be harvested.
b. Products on the market include: insulin, hepatitis B vaccine and human growth
hormone.
2. Transgenic bacteria have been produced to protect and improve the health of plants.
a. Frost-minus bacteria protect the vegetative parts of plants from frost damage.
b. Root-colonizing bacteria receive genes from bacteria with insect toxin, protecting
the roots.
c. Bacteria that colonize corn roots can be endowed with genes for insect toxin.
3. Transgenic bacteria degrade substances.
a. Bacteria selected for ability to degrade oil can be improved by genetic
engineering.
b. Bacteria can be bio-filters to prevent airborne chemical pollutants from being
vented into the air.
c. Bacteria can also remove sulfur from coal before it is burned and help clean up
toxic dumps.
4. Transgenic bacteria produce chemical products.
a. We can manipulate genes coding for enzymes to catalyze synthesis of valuable
chemicals.
b. Phenylalanine used in aspartame sweetener can be grown by engineered
bacteria.
5. Transgenic bacteria process minerals.
a. Many major mining companies already use bacteria to obtain various metals.
b. Genetically engineered "bio-leaching" bacteria extract copper, uranium, gold from
low-grade ore.
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C. Transgenic Plants
1. Plant cells that have had the cell wall removed are called protoplasts.
2. Electric current makes tiny holes in a plasma membrane through which genetic material
enters.
3. Foreign genes now give cotton, corn and potato strains ability to produce insects toxin.
4. Transgenic improvements in wheat and rice will be needed to avoid food shortages in
2020.
5. Stomata leaf openings could be altered to boost CO2 intake or reduce water loss.
6. Plant-produced human hormones would be cheap and lack pathogens that could infect
people.
D. Transgenic Animals
1. Animal use requires methods to insert genes into eggs of animals.
a. It is possible to micro-inject foreign genes into eggs by hand.
b. Vortex mixing places eggs in an agitator with DNA and silicon-carbide needles
that make tiny holes through which the DNA can enter.
c. Using this technique, many types of animal eggs have been injected with bovine
growth hormone (bGH) to produce larger fishes, cows, pigs, rabbits, and sheep.
2. Gene pharming is the use of transgenic farm animals to produce pharmaceuticals; the
product is obtainable from the milk of females.
a. Genes for therapeutic proteins are inserted into animal's DNA; animal's milk
produces proteins.
b. Drugs obtained through gene pharming are planned for the treatment of cystic
fibrosis, cancer, blood diseases, and other disorders.
c. Testing is underway for antithrombin III to prevent blood clots during surgery.
d. Producing the gene product in urine instead of milk uses whole herd from birth
and it is easier to extract proteins from urine.
Gene Therapy
A. Gene Therapy Inserts Healthy Genes
1. This includes procedures to give
patient healthy genes to make up
for a faulty gene.
2. Also includes use of genes to treat
genetic disorders and various
human illnesses.
3. Currently about 1,000 patients
enrolled in 200 gene therapy trials
in U.S.
4. There are ex vivo (outside body)
and in vivo (inside body) methods
of gene therapy.
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B. Genetic Disorders
1. Children with severe combined immunodeficiency syndrome (SCD) underwent ex vivo
gene therapy.
a. Lacking an the enzyme ADA involved in maturation of T and B cells, they faced
life-threatening infections.
b. Bone marrow stem cells are removed, infected with retrovirus that carries a
normal gene, and returned.
c. Use of bone marrow stem cells allows them to divide and produce more cells
with same genes; also can be tested ex vivo to ensure gene transfer has
occurred.
2. Gene therapy trials include treatment of familial hypercholesterolemia where liver cells
lack a receptor for removing cholesterol from blood.
a. High levels of blood cholesterol make patient subject to fatal heart attacks when
young.
b. A small portion of liver is surgically removed and infected with retrovirus with
normal gene for receptor.
c. Chemotherapy in cancer cells kills off healthy as well as cancer cells; genes
given to cancer patients make healthy cells more tolerant or make tumors more
vulnerable.
3. Cystic fibrosis patients lack a gene for trans-membrane chloride ion carriers; patients die
from respiratory tract infections.
a. Liposomes, microscopic vesicles that form when lipoproteins are in solution, are
coated with healthy cystic fibrosis genes and sprayed into a patient's nostrils.
b. Due to limited gene transfer, this method is not yet successful.
4. New research may allow direct correcting of the base sequence of patients with a genetic
disorder.
C. Cancer
1. Chemotherapy for cancer patients often kills healthy cells as well as cancer cells.
a. In an ex vivo trial, bone marrow stem cells from women with ovarian cancer were
infected with a virus carrying a gene to make them more tolerant to
chemotherapy.
b. Another strategy could make cancer cells more vulnerable to chemotherapy.
c. Injecting a retrovirus containing a normal p53 gene into tumors of a lung cancer
patient stopped growth or shrank the tumor; p53 helps regulate cell cycle and
promotes apoptosis in damaged cells.
d. Adenoviruses are easier to use because they can be produced in large quantities
and are active even when not dividing.
e. The virus will kill tumor cells that lack p53 but not healthy cells that have the p53
gene.
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Images: Campbell, Neil and Reece, Jane. Biology (6 ed.) San Francisco: Benjamin Cummings
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Modified Notes: Mader, Sylvia. Biology (7 ed) New York: McGraw Hill Publishing
Notes
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