DNA Technology and
Genomics
History of DNA Technology and Genetic
Engineering
• Genetic Engineering is the process of manipulating
genes and genomes.
• 1953 Watson, Crick, Wilkins and Franklin’s discovery
of the DNA double-helix
• By 2003 the entire human genome had been
sequenced by Craig Venter and the Human Genome
Project, years ahead of schedule.
• But the roots of DNA technology stretch back to the
dawn of history…
Selective breeding
• Since the days that humans settled in the fertile river
valleys, farmers have been trying to perfect crops.
• Selective breeding is the process of choosing
organisms with different desirable traits and mating
them to produce “improved” offspring.
• Black angus beef, Chihuahuas, and bananas are all
examples of selective breeding; over generations,
individuals with a set of characteristics are produced.
Selective Breeding and Polyploidy
• In plants, one way to cram in all of the
desired traits is to combine all of the
parents’ chromosomes.
• So instead of having 2 copies of each
chromosome, some plants have 4, 6, or 8
copies of each chromosome.
• We call species with more than 2 copies
of each chromosome polyploidy.
• Most of our food crops are polyploid,
and most polyploidy organisms are
sterile.
• What are some possible disadvantages?
Genetic Engineering and Recombinant
DNA
• Genetic engineering involves using bacteria like scissors, to cut
sections of DNA, and then gluing in new genes that come from a
different species.
• The DNA that has been artificially made is called recombinant
DNA.
• The product of genetic engineering is a transgenic organism.
• This process is still controversial, with some countries requiring
food products to be labeled GM, or “genetically modified” if they
contain ingredients from a transgenic organism.
• Example: Herbicide tolerance in corn, soybeans, and cotton
Cloning
• Gene cloning is the process by which scientists can produce
multiple copies of specific segments of DNA that they can
then work with in the lab
• Restriction enzymes are used to cut strands of DNA at
specific locations (called restriction sites). They are derived
from bacteria.
• When a DNA molecule is cut by restriction enzymes, the
result will always be a set of restriction fragments, which
will have at least one single-stranded end, called a sticky
end.
• Sticky ends can form hydrogen bonds with complementary
single-stranded pieces of DNA. These unions can be sealed
with enzyme DNA ligase
•
Gene
Cloning
Use of Cloning
• There are two distinct branches of cloning.
• Cell cloning involves inserting a patient’s DNA into an
“adult” cell and growing a tissue or organ in culture.
• This kind of cloning is widespread and has medical
applications from organ transplants, to skin grafts for
burn victims, to neural tissue for muscular dystrophy
sufferers.
• Organism cloning involves putting DNA into an
“embryonic” cell, or stem cell, which could still be any
kind of body part, and then incubating the growing
organism.
• This practice has been going on for years with plants by
“taking cuttings” but only recently in animals, with
Masha the mouse, born the first mammalian clone in
1986.
Cloning a Gene in a Bacterial Plasmid
Finding A Gene of Interest
• Nucleic Acid hybridization is used to find genes of
interest after transformation.
– If we know part of the nucleotide sequence of the gene
we want, we can synthesize a probe complementary to it.
• G-G-C-T-A-A
• Probe: C-C-G-A-T-T
– We now have genomic libraries that log sets of thousands
of recombinant plasmid clones, each of which has a piece
of the original genome being studied
– A cDNA library is made up of complementary DNA made
from mRNA transcribed by reverse transcriptase
Polymerase chain reaction
(PCR)
• Amplification of any piece
of DNA without cells (in
vitro)
• Materials: heat, DNA
polymerase, nucleotides,
single-stranded DNA
primers
• Applications: fossils,
forensics, prenatal
diagnosis, etc.
DNA Technology and Forensics
• CSI and other crime shows, as well as the
paternity tests popular in talk shows, depend on
a process called gel electrophoresis.
• Gel electrophoresis involves running an electrical
current through a gelatin to separate DNA into
bands.
– How does the current help separate?
This DNA has been cut by a restriction
enzyme, creating a kind of DNA
signature.
A DNA Diagnosis
• DNA microarrays, microchips printed with DNA, show
which genes are producing proteins
• These have the potential to change medicine, as they
can show which genes are functioning properly or not
at all and diagnose a disease or disorder years before
it becomes a threat.
Gene Linkage Mapping
• Our first close look at the sequence of DNA was
through gene linkage mapping.
– Gene linkage mapping measure the distance between
genes by the frequency of crossing over moving one to
the homologous chromosome.
– Distant genes are separated by crossing over more often
than nearby genes.
Karyotyping
• A karyotype is a picture of an organism’s chromosomes.
• This is useful in medicine because a karyotype can be
used to detect genetic abnormalities, like duplicate or
fragmented chromosomes.
DNA sequencing and
The Human Genome Project
• After they knew the order of genes, scientists became
curious about the order of nucleotides in these genes,
or gene sequence.
• The first organism to be sequenced was a
bacteriaphage in 1975.
• In 1986, the US Department of Energy announced its
intention to sequence all human genes, also called the
human genome.
• This was formalized in 1990 as the Human Genome
project, a race to sequence all human genes.
Human Genome Project
• In 1990, scientists guessed there were between
50,000 and 100,00 genes in the Human Genome. By
2001, the number had dropped to about 35,000
genes.
• When the project was completed in 2003, 2 years
ahead of schedule, the final tally came to 19,599, far
less than expected.
• Going on complexity alone, humans should have more
genes, C. elegans, one of the simplest animals with
only a few hundred cells has about 20,000 genes.
• Even bacteria have about 5,000 genes on their one
DNA molecule!!
Why So Few?
• Well, we have already learned that not every RNA
codon codes for a different amino acid
– CCA, CCG, CCU, and CCC all call for proline
• Apparently, gene number is not related to how
complex an organism is.
• RNA editing, protein folding, multiple roles of 1
protein, and other factors must account for the
complexity we see.
• So now the new race is to sequence the human
proteome, or all the proteins humans make.
• How can knowing the genome and proteome help
advance medicine?
Bacterial Transformation
Review of the Historical Experiments
Fred Griffith
Avery, MacLeod , McCarty
Fred Griffith 1928
• Studied different strains of Streptococcus
pneumoniae, the bacteria that causes
pneumonia.
• This bacteria come in two strains: S and R
• S-form
– capsule and looks smooth under the microscope.
– Virulent and kills infected mice because the
immune system cannot break through the cell
wall.
• The R-form
– doesn't have a capsule and appears rough.
– The immune system is able to destroy the cell wall.
– This makes the R form non-virulent. The mice
infected by this form of the bacteria will survive.
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Griffith's Experiments
•
•
•
•
Griffith injected mice with the S form
of bacteria and all the mice died from
pneumonia.
He injected mice with the R form of
bacteria and the mice survived the
infection.
He then killed the S form by exposing
them to high temperatures. Mice
injected with these heat-killed bacteria
survived with no ill effects.
He mixed his heat-killed, diseasecausing bacteria with live, harmless
ones and injected the mixture into
mice.
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Griffith's Experiments
• He expected the mice to survive because both strains
were harmless. But, the mice died from
pneumonia!!!
• And he found living S cells in the mice!
• Somehow the heat-killed S strain passed their ability
to cause disease to the live R strain.
• Griffith called this Transformation
–
one strain of bacteria had been changed into another. Some factor
was transferred from heat-killed cell into the live cells. He also
found that the change was permanent.
• He hypothesized that factor could be a gene that
could change the properties of bacteria.
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Frederick Griffith 1928
Transformation of Bacteria
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Avery, MacLeod and McCarty 1944
Avery and his colleagues decided to expand on
Griffith’s experiments to try to identify the
“transforming” material.
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Separated the Components
• Ruptured heat killed S strain bacteria to release their
contents.
• Separated and purified the RNA, DNA, proteins and the
polysaccharide capsules from the bacteria into separate
factions.
•
Mixed each faction with live R bacterial cells and injected them into
mice.
–
–
–
R-bacteria + RNA from S-bacteria = live mouse
R-bacteria + proteins from S-bacteria = live mouse
R-bacteria + polysaccharides from S-bacteria = live mouse
Only R cells were found in their blood.
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Separated the Components
• Ruptured heat killed S strain bacteria to release
their contents.
• Separated and purified the RNA, DNA, proteins
and the polysaccharide capsules from the
bacteria into separate factions.
• Mixed each faction with live R bacterial cells and
injected them into mice.
– R-bacteria + RNA from S-bacteria = live
mouse
– R-bacteria + proteins from S-bacteria = live
mouse
– R-bacteria + polysaccharides from S-bacteria
= live mouse
– Only R cells were found in their blood.
– R bacteria + DNA from S-bacteria = DEAD
MOUSE!!!
– They concluded that DNA was the hereditary
material
Science is full of surprises… be careful with
“no way!”
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

Despite the fact that the Transforming substance
had to be resistant to heat, and proteins are
inactivated by heat…
Most scientists thought that proteins were the
hereditary material because they were more
complex and varied than nucleic acids.
Scientists were generally skeptical, believing DNA
to be too simple a molecule to contain all the
genetic information for an organism.
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