Chapter 7

UNIT 3 Chapter 7: Genetic Research and Biotechnology
Chapter 7: Genetic Research

and Biotechnology
What are some of the risks and benefits of biotechnology?
Although we tend to think of molecular genetics when we
think of modifying organisms, humans have been modifying
organisms for thousands of years through the domestication of
animals and the selective breeding of animals and plants.
These goats have been genetically modified by Canadian

scientists. When these goats mate, their female offspring
will secrete spider silk protein in their milk.
UNIT 3 Chapter 7: Genetic Research and Biotechnology Section 7.1
7.1 Techniques for Producing

and Analyzing DNA
Molecular biology is the study of the

structures and functions of nucleic acids
and proteins.
Recombinant DNA Technology

Recombinant DNA is DNA that has been prepared by
combining fragments of DNA from different sources. These
sources are often genomes of different species.
Constructing recombinant DNA was made possible by the
discovery of restriction enzymes, which help bacteria fend
off viral infections by cleaving viral DNA in the interior of a
molecule.
Molecular biologists are most interested in a special type of
restriction enzyme, called a restriction endonuclease, from
bacteria.
Continued…
• Restriction endonucleases
recognize a specific sequence of
nucleotides, called a target
sequence, within the DNA.
• Next, restriction endonucleases
cut the DNA at a particular point
within the target sequence. This
point is called a restriction site.
EcoRI is a restriction endonuclease that recognizes

and cleaves the sequence GAATTC. The cut DNA
fragment that is produced now has single-stranded
sticky ends at each end of the molecule.
Continued…

Two characteristics that make restriction endonucleases useful to
researchers are:
• Sequence specificity: Cuts made by restriction
endonucleases are specific and predictable.
• Staggered cuts: Most restriction endonucleases produce a

staggered cut that leaves regions, called sticky ends, at the
ends of fragments. Sticky ends can form base pairs with
other single-stranded regions that have a complementary
sequence.
Some restriction endonucleases produce blunt ends. While

this allows any two DNA fragments with blunt ends to
combine, many useless by-products form due to lack of
specificity.
Making a Recombinant
DNA
Molecule
To make a recombinant DNA molecule,
two fragments from different sources are
often cut with the same enzyme to
produce complementary single-stranded
sticky ends.
In this visual, the two fragments are

distinguished by colour. The light-blue
fragment comes from one source, and the
dark-blue fragment comes from another
source.
Base pairing between the complementary

sticky ends brings the molecules together.
DNA ligase then covalently joins the

strands to produce double-stranded
recombinant DNA.
Gene Cloning in Bacteria

Recombinant DNA technology allows scientists to study specific genes
and proteins. This technology is used to produce several identical copies
of a gene in a process called gene cloning.
Gene cloning involves manipulating DNA through many steps to
produce many identical copies of a gene or another segment of DNA in
foreign cells. Scientists use gene cloning to produce sufficient
quantities of a gene or protein for further study.
Bacteria are often used as host systems for gene cloning. They are
inexpensive to maintain and can grow easily in large amounts.
Continued…

• A recombinant DNA molecule composed of the gene of interest
is carried in a vector. For cloning in bacteria, the vector is a
self-replicating, circular piece of DNA called a plasmid.
Continued…

• DNA is taken up by the bacteria through transformation, a
process that involves treating bacteria with chemicals to
make the cell membrane more permeable.
Continued…

• The recombinant DNA is isolated and purified from the bacterial cells.
• The recombinant DNA is analyzed to confirm that the correct
recombinant DNA molecule was made.
The Polymerase Chain Reaction (PCR)

DNA amplification is the process of generating large amounts of
DNA. Gene cloning is one method of DNA amplification.
Another method is the polymerase chain reaction (PCR), which
does not require a host system or recombinant DNA construction.

PCR, which was invented by Kary Mullis,
is an automated process that amplifies specific
regions of DNA from small quantities of
template DNA. Billions of copies of
DNA can be made in a few hours.
Steps in PCR
(1-3)
Continued…
Steps in PCR
(3-4)
Analyzing DNA Fragment Size:

Gel Electrophoresis
Gel electrophoresis is a common
method used to analyze, identify,
and purify DNA fragments. This
method involves using an electric
field to separate DNA fragments
based on their mass and charge.
In gel electrophoresis, the gel lies in an aqueous

buffer solution. A positively charged anode lies at
one end and a negatively charged cathode lies at
the other end. The salts in the buffer carry the
electric charge.
Analyzing DNA Fragment Size:

Gel Electrophoresis
Using a DNA Fingerprint for Identification

DNA fingerprinting analyzes the DNA sequence of certain regions of a
person’s genome. Restriction enzymes and gel electrophoresis can be used to
create a DNA fingerprint (DNA profile). The process can identify a person
because the DNA of an individual is unique (except for identical twins).
In restriction fragment length polymorphism (RFLP) analysis, chromosomal
DNA is treated with restriction endonucleases and analyzed by gel
electrophoresis. The unique band pattern on the gel can be used as a method
of identification if compared to
band patterns from an individual of
known identity.
Using a DNA Fingerprint for Identification

Another method of DNA fingerprinting is short tandem
repeat (STR) profiling. STRs are repeating short sequences of
DNA in the genome that vary in length between individuals.
The length depends on how many copies of a particular STR
are present.
Using primers and PCR, the STRs of an individual are
amplified and then separated by gel electrophoresis. Each STR
fragment is fluorescently labelled, and the fluorescence emitted
can be analyzed by a computer.
DNA fingerprinting using STR profiling produces

a series of peaks that represent STRs of
differing molecular mass and, therefore,
differing lengths. Each individual has a unique
series of
peaks in their STR profile.
Analyzing DNA Sequences

DNA sequencing is a method
for determining, base by base,
the nucleotide sequence of a
fragment of DNA. Originally
developed in the 1970s and
done manually, the process is
now performed by computers.
In 2008, Elaine Mardis published the DNA

sequences of a normal human cell and a
cancerous cell. Next-generation DNA-
sequencing machinery is shown behind her.
Manual DNA Sequencing
In 1977, Frederick Sanger developed

dideoxy sequencing. This sequencing
method uses DNA polymerase to
synthesize DNA fragments of
different lengths from the DNA of
interest. The different-sized fragments
Dideoxynucleotides lack a –OH
group at the 29 and 39 carbons on occur due to the incorporation of one
the ribose sugar. As a result, DNA
synthesis terminates when one of
of four dideoxynucleotides into a
four possible dideoxynucleotides DNA strand during replication, which
are incorporated.
terminates the process.
Steps in Dideoxy Sequencing

1. The DNA to be sequenced is denatured,
and a primer anneals (attaches itself
through base pairing) to the 3′ end of
the region to be sequenced.
2. Four separate reaction tubes are
prepared. Each contains:
• denatured DNA with primer
• deoxynucleotides
• DNA polymerase
• plus one of the four
dideoxynucleotides (ddG, ddC,
ddA, or ddT)
3. DNA synthesis proceeds

in each reaction tube.
DNA fragments of
different lengths are
produced after the
incorporation of
dideoxynucleotides.
4. Each reaction tube’s
fragments are separated
using gel electrophoresis.
Continued…

5. The gel is read from top to bottom. Because the final base
of each fragment and the length of each fragment are
known, the sequence can be determined.
6. The sequence read from

the gel is the synthesized
strand. The sequence of
the original template DNA
is complementary to this
sequence.
Automated DNA Sequencing

Early Automated DNA Sequencing
The Human Genome Project was made possible by the
development of early automated DNA sequencing techniques.
The project involved sequencing the human genome and
identifying the genes within it. Early automated sequencing
techniques were quicker and more efficient than manual
sequencing because they used computer technology.

Next-generation DNA Sequencing
Dideoxy sequencing has been replaced by “next-generation”
methods that provide even faster sequencing output. The
Human Genome Project took ten years to complete; now, a
human genome can be sequenced in less than nine months.
Making Sequence-Specific Mutations

In 1976, Canadian scientist Michael Smith developed a method
called site-directed mutagenesis to study human-made mutations
and their cellular effects.

Site-directed mutagenesis is a
method of specifically altering the
nucleotide sequence of a
region of DNA. This allows
scientists to study the
structure and function
of genes and proteins.
The Michael Smith Laboratories at the University of

British Columbia are named after the Nobel laureate.
7.2 Production and Regulation of

Genetically Engineered Organisms
Gene therapy treats genetic disorders by introducing a correct
form of the disease-related gene into the patient’s genome. The
result produces a recombinant human.
This technique has been used to treat diseases such as Duchenne
muscular dystrophy.
Before gene therapy (left), the muscle
fibres of the mouse do not produce
enough dystrophin, a protein required
for normal muscle function. The orange
and yellow portions of the tissue
represent dead cells.
After gene therapy (right), dystrophin

levels are normal and the muscle fibres
are healthy.
Production and Regulation of Genetically

Engineered Organisms
Recombinant DNA techniques have also been used to create
genetically modified organisms (GMOs) such as GloFish®, which are
sold in some countries as novelty pets. The sale of GloFish® is banned
in Canada because of ethical concerns arising from the technology.

Concerns are related to both
scientific boundaries (“How
far should we go?”) and
regulations (“Who should
make these decisions?”).
Applications of Genetically
Engineered Organisms
The techniques discussed in this chapter involve the alteration of
the genetic material of an organism in a specific manner.
Collectively they are known as genetic engineering. These
techniques can also include the introduction of foreign DNA into
an organism’s genome, which results in a transgenic organism (a
new phenotype). Transgenic organisms are often referred to as
genetically modified organisms or GMOs.
The term biotechnology refers to
the use of the tools of molecular
genetics to produce such Although these Europigs™ look the same as
organisms and their products. non-GM pigs, their excrement contains less
phosphorus—a substance that pollutes aquatic
ecosystems. Is this a good idea? Why? Why not?
Applications of Transgenic Bacteria in

Pharmaceuticals
A transgenic organism such as bacteria can be used to produce
large amounts of medicines such as human insulin. In this
process, recombinant DNA technology is used to produce
transgenic bacteria. An expression vector is used as a plasmid.
This vector includes sequences that support transcription and
translation, which allows for the production of a pharmaceutical
protein when the vector is transformed into bacteria.
In 1993, Health Canada approved the sale of human insulin
produced by transgenic bacteria. Previously, insulin was purified
from animals and produced many adverse side effects in patients.
These side effects are not present when patients use human
insulin produced in bacteria.
Insulin Production by Transgenic Bacteria

Bacteria can be engineered to express large
quantities of a protein such as human insulin.
Transgenic Bacteria and Bioremediation

Another application of transgenic organisms is bioremediation, which
involves using micro-organisms to reduce environmental pollutants.
Many of these micro-organisms are genetically modified to improve their
ability to convert pollutants
into non-toxic products.

Dr. Ananda Chakrabarty

developed transgenic bacteria that could break down crude oil
faster than naturally occurring bacteria. He was awarded the first patent
for a recombinant organism, opening the doors for other biotechnology
patent applications and approvals.

Transgenic Plants
Agricultural plants are the most common examples of GMOs and include
herbicide- and disease-resistant corn, soybeans, canola, tomatoes, and
potatoes. Some transgenic plants are also used to mass-produce medicines.
Two methods to introduce foreign DNA into plant cells to produce
transgenic plants are:
• biolistic method: plants cells are bombarded with particles

coated with DNA that integrate into the genome. This
method cannot control the DNA insertion site or
quantities.
• Ti plasmid method: a tumour-inducing (Ti) plasmid is
used as a vector for inserting DNA into the plant genome.
Producing Transgenic Plants Using the

Ti Plasmid Method
The same basic principles that are

used to make transgenic bacteria can
be applied to plants.
In the case of transgenic plants, the

vector used to carry the new gene is
the Ti plasmid from Agrobacterium
tumefaciens.
Continued…
Producing Transgenic Plants Using the

Ti Plasmid Method
The Controversy Surrounding

Transgenic Plants
Are genetically modified crops safe for human consumption?
• In Canada, GMO crops require approval by

Health Canada. This process takes 7 to 10 years.
• Food safety issues, such as the potential for
introducing new toxins and allergens, are
potential risk factors.
• Long-term side effects are unknown.
Continued…
The Controversy Surrounding

Transgenic Plants
Will genetically modified crops have a negative impact on the environment?
• Gene transfer, for example transferring

traits such as antibiotic resistance to
other organisms, could occur.
• Toxins produced by transgenic plants
to protect them from insects may
affect non-target organisms.
Golden rice is a GMO that contains carotenoids

needed for Vitamin A synthesis.
Transgenic Animals and Related

Controversies
Producing transgenic animals involves inserting a foreign
gene into the genome of an animal oocyte (egg) that is then
fertilized. The fertilized egg is implanted into a host
female, and the resulting offspring are the transgenic form
of the animal.
Gene pharming is the process of using transgenic animals
to produce therapeutic proteins. These proteins are targeted
for production in the mammary glands of a transgenic
animal, which allows them to be easily purified from the
milk.
Transgenic Animals: Gene Pharming

Mammalian Cloning from Somatic Cells

The procedure for cloning mammals
was first successful in 1996 with the
cloning of a sheep named Dolly by
scientist Ian Wilmut and his
colleagues. Since then, other animals
such as cows, pigs, mice, dogs, and
cats have been cloned.
Cloning mammals involves fusing a donor somatic cell with an

oocyte that has had the nucleus removed. The resulting embryo
is implanted in a host. The offspring is genetically identical to
the animal that was the source of the somatic cell.
Continued…
Mammalian Cloning from Somatic Cells

Controversies Surrounding
Transgenic Animals
Is the cloning of livestock and pets for the benefit of humans ethical?
• Applications (such as gene pharming)

continue to be pursued The first pet to be cloned was a cat
named CC (for carbon copy).
Is human cloning something that

should be studied?
• Numerous moral, ethical, and
legal debates surround the
idea of human cloning.

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Chapter 7

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UNIT 3 Chapter 7: Genetic Research and Biotechnology

Chapter 7: Genetic Research

These goats have been genetically modified by Canadian

7.1 Techniques for Producing

Molecular biology is the study of the

Recombinant DNA Technology

Recombinant DNA Technology

EcoRI is a restriction endonuclease that recognizes

Recombinant DNA Technology

• Staggered cuts: Most restriction endonucleases produce a

Some restriction endonucleases produce blunt ends. While

In this visual, the two fragments are

Base pairing between the complementary

DNA ligase then covalently joins the

Gene Cloning in Bacteria

Gene Cloning in Bacteria

Gene Cloning in Bacteria

Gene Cloning in Bacteria

The Polymerase Chain Reaction (PCR)

Analyzing DNA Fragment Size:

In gel electrophoresis, the gel lies in an aqueous

Analyzing DNA Fragment Size:

Using a DNA Fingerprint for Identification

Using a DNA Fingerprint for Identification

DNA fingerprinting using STR profiling produces

Analyzing DNA Sequences

In 2008, Elaine Mardis published the DNA

Manual DNA Sequencing

In 1977, Frederick Sanger developed

Steps in Dideoxy Sequencing

Steps in Dideoxy Sequencing

3. DNA synthesis proceeds

Steps in Dideoxy Sequencing

6. The sequence read from

Automated DNA Sequencing

Making Sequence-Specific Mutations

The Michael Smith Laboratories at the University of

7.2 Production and Regulation of

After gene therapy (right), dystrophin

Production and Regulation of Genetically

Applications of Transgenic Bacteria in

Insulin Production by Transgenic Bacteria

Transgenic Bacteria and Bioremediation

Dr. Ananda Chakrabarty

• biolistic method: plants cells are bombarded with particles

Producing Transgenic Plants Using the

The same basic principles that are

In the case of transgenic plants, the

Producing Transgenic Plants Using the

The Controversy Surrounding

• In Canada, GMO crops require approval by

The Controversy Surrounding

• Gene transfer, for example transferring

Golden rice is a GMO that contains carotenoids

Transgenic Animals and Related

Transgenic Animals: Gene Pharming

Mammalian Cloning from Somatic Cells

Cloning mammals involves fusing a donor somatic cell with an

Mammalian Cloning from Somatic Cells

• Applications (such as gene pharming)

Is human cloning something that

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