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DNA Technology
Genetic Engineering
• Alteration of genomes for medical or
industrial purposes.
– Can be used in bacteria, plant, and animal cells
The Cloning of a Gene
• Cloning
– The production of identical copies through some
asexual means.
– All human identical twins
• Gene Cloning
– The production of many identical copies of a
single gene.
The Cloning of a Gene
• Why?
– Determine the difference between normal and
mutated genes
– Alter the phenotype of organisms
• Called gene therapy in humans
Recombinant DNA Technology
• Contains DNA from two or more sources
– Ex. human and bacteria
• Vector: a piece of DNA that can be
manipulated, in order to add foreign DNA to it
– Plasmids: small accessory rings of DNA from
bacteria that are not part of the bacterial
chromosome and are capable of replicating on
their own. Most common in bacteria
Recombinant DNA Technology
• Using Recombinant DNA Technology (Figure 24.15)
– Restriction enzymes cleave (break open) a plasmid vector
at specific site. Foreign DNA that is to be inserted is also
cleaved with same restriction enzyme
– The single-stranded, but complementary, ends of the two
DNA molecules are called “sticky ends”.
– Foreign DNA is inserted into plasmid DNA and “sticky
ends” pair up
– DNA ligase seals them together
– Bacterial cells take up the recombinant plasmids. As
plasmid replicates, the gene is cloned.
Recombinant DNA Technology
Polymerase Chain Reaction
• Can create millions of copies of a segment of DNA
– quickly in a test tube
– No vector or host cell
• It amplifies (makes copies of) a targeted DNA
sequence
• Requires DNA polymerase, a set of primers, and a
supply of nucleotides
– Primers are single stranded DNA sequences that start
replication
• Amount of DNA doubles with each replication
Polymerase Chain Reaction
• During the PCR cycle, the mixture of DNA, DNA
polymerase, two primers, and nucleotides is
heated to 95°C to separate the two strands of the
double helix
– The DNA polymerase was extracted from the
bacterium Thermus aquaticus, which lives in hot
springs
• The temperature is lowered so the primers pair
with the DNA strands
• DNA polymerase copies the DNA between the
primers
• At the end of the second cycle there are 4 copies,
after 25-30 cycles there are over 30 million copies
DNA Fingerprinting
• Permits identification of individuals and their
relatives.
• Based on differences in the sequence of DNA
nucleotides between individuals.
DNA Fingerprinting
Medical use: identify the presence of a viral
infection or mutated gene.
Forensics: DNA from a crime scene can identify
the suspect.
Identify people: can identify the parents of a
child or the identity of body remains.
Biotechnology
• Uses natural biological systems to create a
product or to achieve an end desired by
human beings.
– Bacteria, plants, and animals
• Transgenic organisms: organisms that have
had a foreign gene inserted into them.
Transgenic Bacteria
• Recombinant DNA technology is used to
produce transgenic bacteria.
• Medical Uses: insulin, human growth
hormone, hepatitis B vaccine
• Agricultural Uses: bacteria that protect plants
from freezing, bacteria that protect plant
roots from insects.
• Environmental: bacteria that degrade oil,
bacteria that remove sulfur from coal
Transgenic Plants
• Foreign genes are introduced into plant
embryos or protoplasts
– When protoplasts are suspended in a liquid
containing foreign DNA, an electric current makes
tiny holes in the plasma membrane where genetic
material can enter.
• Protoplasts develop into mature plants
containing and expressing the foreign DNA
Transgenic Plants
• Makes plants that are resistant to herbicides
(soybeans), plants that secrete toxins that kill
insects (cotton, corn, potato), some are both
• Plant Pharming: the use of engineered plants
to produce pharmaceuticals
– Golden rice: designed to be grown and consumed
in areas with a shortage of dietary vitamin A, a
deficiency which is estimated to kill 670,000
children under the age of 5 each year.
Transgenic Animals
• Genes are inserted into the eggs of animals.
• By hand or vortex mixing
– eggs placed in a agitator with DNA and silicon-carbide
needles.
– The needles make holes in the eggs where the DNA
can enter.
– When the eggs are fertilized, the offspring are
transgenic animals.
• Bovine growth hormone: used to make larger
fishes, cows, pigs, rabbits, and sheep
Transgenic Animals
• Animal pharming: the use of transgenic farm
animals to produce pharmaceuticals
– DNA containing the gene of interest is injected
into donor eggs.
– Following fertilization in the lab, the zygotes are
placed in host females, where they develop
– The offspring will produce the biotechnology
product
• Drugs in the milk of goats
DNA Fingerprinting
• Restriction enzymes cut DNA into fragments
• Fragments are separated by gel electrophoresis,
– DNA fragments migrate though a gel according to their
length when an electric field is applied.
– The shorter the fragment, the farther it migrates
– Carried out by detecting how many times a short sequence
is repeated
• People differ by how many repeats they have at
particular locations
– PCR amplifies only particular portions of the DNA. The
greater the number of repeats, the greater the amount of
DNA that is amplified by PCR. The