CO QAH + MELC LW

Course Outline & Quality Assured HANDOUT No. 1

Handouts paired with MELC- Based in GENERAL BIOLOGY 2
Learner’s Worksheet

MELC:
1. Outline the process involved in genetic engineering. (STEM_BIO11/12-IIIa-b-6)
2. Discuss the applications of recombinant DNA. (STEM_BIO11/12-IIIa-b-7)
Semester: 1 Week No.1 Day: 1 - 5

According to National Human Genome Research Instituteꓹ genetic engineering is the process of using
recombinant DNA (rDNA) technology to alter the genetic makeup of an organism. Traditionallyꓹ humans have
manipulated genomes indirectly by controlling breeding and selecting offspring with desired traits. They also
added that genetic engineering involves the direct manipulation of one or more genes. Most often the gene
from another species is added to an organism’s genome to give it a desired phenotype.

LESSON 1: GENETIC ENGINEERING

Genetic engineering is the process of using recombinant DNA (Rdna) technology to alter the genetic
makeup of an organism. Traditionally, humans have manipulated genomes indirectly by controlling breeding
and selecting offspring with desired traits. Genetic engineering involves direct manipulation of one or more
genes. Most often, a gene from another species is added to an organism’s genome to give the desired
phenotype.
Commonly used methods of genetic engineering includes:
- Microinjection of DNA into the nucleus of anchored cells;
- Electroporation, where DNA is introduced through cell membrane pores by pulsed electrical
charges;
- Polycationic neutralization of the cell membrane and the DNA to be introduced to improve passive
uptake.

When one thinks of genetic engineering, they are more likely to picture a complicated science
which involves altering the very building blocks of life. The are several steps in the process of
genetic engineering. Scientist follow a step by step process in order to alter the DNA of an organism.
In describing the process, the example of a soybean will be used as a guide to each step.

1. First, a gene is picked that will be altered, added or removed. This step requires that the
wanted gene is found and isolated. If a new breed of soybean were to be given a
pesticide, the effective bacteria in the pesticide would be isolated. The bacteria genes are
needed in order to be placed in the soybeans genome.
2. The isolated gene is copied several times.The DNA from the bacteria is then copied
several times. This is done by splitting the DNA down the center of the double helix and
pairing it with the appropriate chemical (Goldbas).
3. The gene is transferred to the new organism. It is transferred into the tissue of the
organism. Since it is impossible to insert the DNA into each cell of an organism, the DNA
is now injected into the tissue of the soybean plant. The soybean plant is then grown to
maturity in a greenhouse. A technique used today involves shooting DNA from a .22
caliber charge into plant tissue (Goldsburg).
4. Create a new plant/animal/trait from the newly modified tissue. Now that the new DNA is
present, the soybeans will continue to grow, only now they posses a new trait.
5. Check the insertion produces desired results.
6. Check that the new gene can be found in the offspring (seeds) of the organism. This is
most crucial step in genetic engineering. If the offspring of the genetically modified
organism does not possess the traits given to the parent, the engineering has failed and
must be done again.

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 LESSON 2: RECOMBINANT DNA

Brief History of recombinant DNA Technology

In the late 1960’s Stewart Lim and Werner Arber discovered restrictions enzymes in E.coli,
which are known as endonuclease. Endonuclease cut DNA at specific site where there are adjacent
base sequence. They also create sticky ends on the cut DNA sites, allowing certain DNA fragments
to be joined together. The use of such endonuclease in modifying DNA. However, was not discovered
until 1973, when Herbert Boyer and Stanley Cohen performed a successful set of experiments. The
first one involves the recombination of plasmids in the DNA of E.coli bacterium. These plasmids
contain genes that code for resistance to certain antibiotics. The next involves recombining plasmid
DNA of the bacterium into fragments of frog DNA. The recombined plasmid resulted in the production
of an extra protein in the bacterium. Therefore, the frog gene can be successfully expressed in the E.
coli bacterium.
The success of these experiments was revolutionary because scientists can now manipulate
genes create organisms with favorable traits. However, a year later, fueled by the growing
communication and fear among the general public, scientists led by Paul Berg wrote a letter to the
National Academy of Sciences, to express their concerns about the possible dangers and ethical
considerations involved in gene manipulation. Thus, Asilomar Conference was held in February 1975
to discuss and help establish the proper guidelines in using recombinant DNA.
Before we discuss the several techniques involved in recombinant DNA technology, we must
first learn about the different tools used in this process.
Technologies and Tools Used in Recombinant DNA Technology
Molecular Biologist have developed different technologies and tools that allows them to study
and manipulate DNA molecules. The process that will be discussed here are gel electrophoresis, DNA
splicing, and polymerase chain reactions.

Gel Electrophoresis
Gel Electrophoresis is a method used to separate DNA fragments based on their size. In this
method, a mixture of DNA Fragments is placed at one end of a porous gel, and an electric voltage is
applied to the gel. The negatively charged DNA molecules move toward the positive end of the gel.
The smaller the DNA fragments, the faster they move. This is important for characterizing DNA
fragments, fingerprinting, comparing the genome of different organism, and locating and identifying
one particular gene out of the millions of genes in an individual’s genome.

Fig.1 Apparatus used in gel electrophoresis

DNA Sequencing
This is a method used to provide the identity and order of nucleotides in a DNA strand. Small,
single-stranded pieces of DNA are placed in test tubes with an enzyme that can make a
complementary DNA strand by using the original DNA strand as a template. A supply of the four
nucleotide bases found in DNA is then added, along with a small amount of one of the bases that has
been labeled with fluorescent dyes.

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 Fig. 2 Process involved in DNA sequencing

Polymerase Chain Reaction (PCR)

The goal of PCR is to amplify specific DNA sequences. This is important in detecting diseases
or infectious agents. To make copies of a piece of DNA. DNA is heated to separate its two strands
then cooled to allow the primers to bind to the single-stranded DNA. The priers are short DNA strands
that provide a place for the DNA polymerase to start working. As the polymerase starts working, new
strands of the separated DNA are formed. Continuous heating and cooling allow further separation of
DNA and formation of new DNA strands, respectively, creating millions of copies of the DNA segments.

Fig.3 Results of polymerase chain reaction (PCR)

Process Used in Recombinant DNA Technology

There are three methods by which recombinant DNA is made. These are transformation,
vectorless gene transfer, and transduction.
BIG IDEA: Recombinant DNA technology has proven to be useful in various fields such as medicine
and agriculture.

Transformation Using a Vector

Recombinant DNA may be created through transformation with the help of a vector such as
bacteria cells. Vectors are organisms that are normally harmless but may help spread infection by
transferring the genetic material from one host to another. In the transformation process, a selected
portion of the foreign DNA is inserted into a small, circular DNA molecule called Plasmid, which is
naturally found in bacteria. Plasmids are the most useful tool in gene transformation for two reason.
First, a plasmid contains a gene sequence that serves as a bacteria origin or replication. This is where
the foreign DNA can be inserted into the bacteria cell. Second, it also contains a genetic marker,
which makes it possible to distinguish bacteria that carry plasmids-containing foreign DNA. Some of
these genetic markers code for antibiotic resistance.
During transformation, a restriction endonuclease enzyme is used to cut the piece of the
door DNA. This enzyme cleaves the DNA at the phosphate-sugar bond, and thus sticky ends are
created. Sticky ends are areas in the DNA where the bases are ready to paired. Restriction enzymes
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cut the DNA only at specific nucleotides sequence. They work precisely like a key that fits only one
specific lock. Then, an enzyme known as DNA ligase is used to insert the donor DNA into the vector.
It seals the sticky ends by joining the phosphate and the sugar bonds in the DNA. The inserted DNA
also contains a genetic marker for identification. The recombinant DNA is then inserted into a bacterial
cell, such as E.coli.
After transformation, the culture is treated with an antibiotic. Those that have been
transformed will be the only one to survive because they can carry the resistance gene.

Fig. 4 Summary of DNA recombinant technology

Vector Gene Transfer

The process is similar to transformation, but it does not involve vectors. The type of vectorless
gene transfer include electroporation, protoplast fusion, microinjection, and use of particle gun. In
electroporation, temporary holes are formed in the plasma membrane of host cell by applying a
significant amount of electricity in the culture medium. This enables the entry of foreign DNA via the
pores. In protoplast fusion, cells are treated with chemicals to initiate recombination. In this process,
bacteria cell walls are digested, turning the cells into protoplasts. These protoplasts are treated with
polyethylene glycol to allow them to fuse, creating a random recombination of genes. The resulting
recombinant cell will now grow a new cell wall.

Fig. 5 Process involved in protoplast fusion

In microinjection, the host cell is immobilized by applying a mild suction with blunt pipette. The foreign
gene is then injected with a microinjection needle, thus creating recombinant DNA.

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 Fig. 6 Microinjection is commonly used in invitro fertilization. In this picture, the DNA from sperm
cell is injected into the egg cell.

Using a particle gun for recombination, the host cell is bombarded with tungsten particles
coated with foreign DNA. This process is used in the field of agriculture. Many farmers use this method
to genetically modify plants to make them highly resistant to insects and other pests. Some also use
this method to develop crops that can survive extreme weather condition.

Fig. 7 Comparison between vector DNA technology and the particle gun method adapted

Transduction

Transduction is the process

wherein genetically engineered
bacteriophages- viruses that that
parasitize bacteria- are introduced into the
cell to create the desired recombinant
DNA. The steps involved in transduction
are shown in figure 8.

Fig. 8 Steps involved in transduction

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Application of Recombinant DNA

Several scientific advancements have led to many genetic engineering techniques that are
very beneficial to us. It is now possible to transfer DNA sequences from one organism to another.
American researcher Steven Howell and his associates at the University of California in San Diego
learned that even genes from two or more different organisms can be made to work together. Howell’s
team tried to isolate the gene to luciferase- an enzyme that allows fireflies to glow- and insert it into
tobacco cells.

Fig. 9 A glow-in-the-dark tobacco plant

When the gene was activated from the recombinant cells, the plants glowed in the dark. This
means that the basic mechanisms of gene expression are shared by both plants and animals. Here
are some of the benefits that we can get from recombinant DNA.

Agriculture
Transgenic plants, or plants that contain genes from other organisms are now important
part in the field of agriculture. By using recombinant DNA technology, plants can be grown with genes
responsible for producing natural insecticides. This technology reduces the need for synthetic or
artificial insecticides and pesticides. In the Philippines, recent developments have enabled plants to
express a recombinant form of protein used by the Bacillus thuringiensis bacterium. Recall the
genetically modified maize known as Bt corn. This crop is widely used in organic farming because it
is poisonous to certain pests. This is greatly beneficial to farmers because Bt corn eliminates corn
borers, which cause an enormous amount of damage to corn crops in our country.

The following are some bacteria used in

recombinant DNA technology:
1. Pseudomonas syringae – The recombinant
variant of this bacterium is called the ice-minus
bacterium, which lacks the gene responsible for
ice formation. The ice-minus bacteria prevent
frost crystals from forming on plants.
2. Pseudomonas flourescens- This is a
nonpathogenic bacterium that has the ability to
produce proteins rapidly. This characteristic is
advantageous in developing biotherapeutics
and vaccines.
3. Agrobacterium tumefaciens- In its natural state,
this bacterium has a tumor-inducing (Ti)
plasmid that causes crown gall disease in
plants. The said Ti plasmid in the bacterium can
be removed and replaced with a recombinant
plasmid. This enables the now-modified Fig. 10 Steps in growing Bt corn
bacterium to introduce beneficial genes to
plants.

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Medicine
An important contribution of recombinant DNA technology in the field of medicine is the use
of bacteria to create substances that our body needs, whether to maintain good health or to treat a
disease. For example, human insulin is created using recombinant DNA to help diabetic. Also, the
modified human growth hormone is widely used to support the development of people who have a
malfunctioning pituitary gland. This is of great benefit because it avoids the practice of getting growth
hormones from dead bodies, which can pose serious health risks. Other important substances such
as blood clotting factors (for people suffering from hemophilia) and hepatitis B virus surface antigens
(for people suffering from hepatitis B) are also products of recombinant DNA technology. Certainly,
this technology has given us many benefits in the field of medicine.

Fig.11 How recombinant DNA technology is used in medicine

Food Industry

Recombinant DNA has also improved the food industry. Some of the crops that we eat are
now resistant to pests, disease, and environmental stress. As a result, crop yields have increased and
production costs have been kept lowered. Recombinant crops also beneficial because of their
improved nutritional quality and longer shelf life.
BIG IDEA: Genetically modified food is a widely debated application of recombinant DNA technology.
However, it is really obvious that this technology has been beneficial to both producers and consumers
of agriculture products.
Recombinant technology can also be used for processing high-quality fermented food such as cheese,
pickles, wines and beers.

Fig. 12 Recombinant DNA technology is responsible in creating genetically modified organisms.

Some of these GMO’s serve as food. This photo shows the comparison between the borer-affected
corn crops (Right) and the Bt corn crops (Left).

REFERENCES
“Genetic Engineering.” Genome.gov. Accessed Julty 08ꓹ2021. https://www.genome.gov/genetics-
glossary/Genetic-Engineering

"The Process of Genetic Engineering." The Basics of Genetic Engineering. Accessed July 08, 2021.
https://sites.psu.edu/english202geneticengineering/genetic-engineering/how-it-works/.

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 Prepared by: LADY DARYL A. BERBON
SPNHS