Processes of Genetic Engineering

Learning Competency:

Outline the processes involved in genetic engineering (STEM_BIO11/12-IIIa-b-6).

Key Concepts

The wide variety of plant-based products and fortified formulas in the market today has
been made possible by artificial selection of desired genes through genetic engineering - a gene
modification process wherein the Deoxyribonucleic acid (DNA) is transferred from one organism to
another.
Classical breeding focuses on the mating of organisms with desirable qualities or traits
while genetic engineering involves molecular techniques to modify the traits of a target organism.
The modification of traits may involve the introduction of new traits into an organism or
enhancement of a present trait by increasing or disrupting the expression of the desired gene. It
has an application in the pharmaceutical, industrial, agricultural, medical, and other industries.
Genetically modified organisms have been subject for public scrutiny whether it is safe to use or
ethically accepted. These challenge the researchers to prove the significance of GMOs as a
breakthrough in science.

Processes of Genetic Engineering

Stage 1. DNA Cleavage - A restriction endonuclease is used to cleave the source DNA into a different
set of fragments. The endonuclease’s recognition sequence is likely to occur many times within
the source of DNA, thus cleavage will produce a large number of different fragments. The
fragments can be separated from one another according to their size by gel electrophoresis.
Stage 2. Production of Recombinant DNA - The fragments of DNA are inserted into plasmids or
viral vectors that have been cleaved with the same restriction endonuclease as the source
DNA.
Stage 3. Cloning - The plasmids or viruses serve as vectors that can introduce the DNA fragments
into cells--- usually, but not always bacteria. As each cell produces, it forms a clone of cells
that all contain the fragment-bearing vector.
Stage 4. Screening - The clones containing a specific DNA fragment of interest are identified from
the clone library.

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 Figure 1. Processes of Genetic Engineering
Source: Raven, Peter H. et al. Biology. 2005

Methods of Introducing Plasmids into the Host Organism

1. Biolistics - This technique uses a “gene gun” to fire

DNA-coated pellets on plant tissues. Cells that are
able to survive and take up the expression plasmid
coated pellets can acquire the ability to express the
designed protein.

Figure 2. Biolistics
Source:https://commons.wikimedia.org/w/index.php?curid=40535573

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 2. Plasmid insertion by Heat Shock Treatment - is a process used to transfer plasmid DNA into
bacteria. The target cells undergo a pretreatment procedure to increase the pore sizes of
their plasma membranes. The pretreatment using Calcium chloride makes the cells
“competent” for the introduction of the plasmid DNA, then the cells are incubated with the
desired plasmid at about 4°C for about 30 minutes. During this time, the plasmids
concentrate near the cells. Afterward, a “Heat Shock” is done on the plasmid-cell solution
by incubating it at 42°C for 1
minute then back to 4°C for 2
minutes. The rapid rise and
drop of temperature increase
and decrease the pore sizes in
the membrane, respectively.
The plasmid DNA near the
membrane surface is taken
into the cells by this process.
The cells that took up the
plasmids acquire new traits
and are called “transformed
bacterium”.
Figure 3. Heat Shock Treatment
Source:https://www.khanacademy.org/science/biology/biotech-dna-
technology/dna-cloning-tutorial/a/bacterial-transformation-selection

3. Electroporation - This technique

follows a similar methodology as
Heat Shock Treatment but uses
electric shock to expand the
membrane pores. This method is
commonly used for the insertion
of genes into mammalian cells.

Figure 4. Electroporation
Source: https://www.btxonline.com/technical-resources/faq.html

Methods to Screen Recombinant Cells

1. Selection of plasmid DNA containing cells - A selection marker within the inserted
plasmid DNA sequence allows the selection of “transformants”. Usually, an antibiotic
resistance gene is included in the plasmid DNA. This mechanism allows only “transformed”
cells to survive in the presence of the antibiotic (e.g. ampicillin).

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 Figure 5. Selection of plasmid DNA containing cells
Source: Raven, Peter H. et al. Biology. 2005

2. Selection of transformed cells with the desired gene - The most general procedure for
screening clone libraries to find a particular gene is hybridization - the cloned genes form
base-pairs with complementary sequences on another nucleic acid. The complementary
nucleic acid is called as probe because it is used to probe for the presence of the gene of
interest. In this method of screening, bacterial colonies, forming a replica of the plate. The
filter is then treated with a solution that denatures the bacterial DNA and contains a
radioactively labeled probe. The probe hybridizes with complementary single-stranded
sequences on the bacterial DNA. The filter is laid over photographic film and areas that
contain radioactivity will expose the film( autoradiography). Only colonies that contain the
gene of interest hybridize with the radioactive probe and emit radioactivity onto the film.
The pattern on the film is then compared to the original master plate to identify the gene-
containing colonies.

Figure 6. Hybridization
Source: Raven, Peter H. et al. Biology. 2005

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 3. Polymerase Chain Reaction (PCR) detection of plasmid DNA- the presence of the desired gene
in the inserted plasmids may also be confirmed through PCR amplification. PCR reactions
specific for the desired gene may be done using DNA from cells that would confirm the
presence of the gene within the samples. PCR reactions specific for plasmid sequences will
confirm/identify the type of plasmid used for the transformation through the following steps:
Step 1. Denaturation. An excess primer, synthetic sequence of 20 to 30 nucleotides.
is mixed with the DNA fragments to be amplified. These mixture of primer and
fragments is heated to about 98°C in order to dissociate the double-stranded
DNA fragments into single strands.
Step 2. Annealing of Primers. The solution is allowed to cool to about 60°C so that the
single strands of DNA reassociate into double strands.
Step 3. Elongation. Using the primer, the polymerase copies the rest of the fragment
and both DNA strands are replicated resulting into two copies of the original
fragment.

Figure 7. PCR Amplification

Source: https://benchling.com/s/prt-0vlAQwMiXl1ivfOdmoHT/edit

Despite the proposed benefits of GMOs, some people are concerned regarding the
consumption of these modified foods. While most of the products are tested for safety, concerns
are raised for the possibility of not being able to detect hazards that are present but are currently
undetectable by today’s current technology. Because of these issues, manufacturers are urged to
provide labels that inform consumers of the presence of GMOs in their products. While GMOs are
believed to be safe when licensed by the food regulatory agencies, the consumers must be provided
with enough information to make their own choices regarding their use.

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