General Biology II

Week IA: Processes of Genetic Engineering

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.
 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.

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”

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.

 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 aortic (e.g., ampicillin).

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 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.
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.

Week IB: The Applications of Recombinant Deoxyribonucleic Acid (rDNA)

What is Recombination?
- a process of forming a new combination of genes by rearranging genetic material.

It can occur naturally by:

a. crossing over in chromosomes during meiosis.

b. union of genes from male and female parents during sexual
reproduction.
c. in all types of genetic exchange in prokaryotes.

The method can also be done artificially by:

- joining segments of DNA from different organisms - as a result of genetic engineering.

- This means that there is human intervention in the process of combining genes to manipulating the
molecular basis of inheritance is collectively called Recombinant DNA Technology or Biotechnology
(Audesirk and Audesirk).

What is Recombinant DNA?

- Recombinant DNA or rDNA is a molecule of DNA that has been altered or changed, either through a
natural process or through laboratory techniques.
- In the perspective of biotechnology, rDNA is the artificial or uncommon union of DNA fragments from
two different sources of genetic material.

The organism that serves as the source of the desired DNA section is called the donor, while the organism whose
DNA is modified is called the vector.

Some scientists also use the term chimeric DNA for

this unnatural combination of genes.
The human cell serves as the donor since the
desired gene for human growth hormone is clipped
from it,
while the bacterium is the vector as its DNA is the one being modified.

The resulting new DNA is the rDNA which will be inserted to a living host cell in order to produce the desired
human growth hormone.

The experimental manipulation of genetic materials to produce rDNA is what is now called recombinant DNA
technology (Schlichte).

The resulting organism that carries the transgene or the artificially inserted gene is called a transgenic organism or
a genetically modified organism or GMO.

Table I. Applications and Products of Recombinant DNA Technology

Application
in Different Products What is it?
Fields
A. Medicine a) Insulin It is a hormone made up of protein that is secreted in the pancreas
and Health by islet cells; responsible for controlling glucose (sugar) level in
humans. If a person has low amount of Insulin in his body, he
will suffer from a disease called diabetes. Nowadays, human
insulin is readily available in the market. Synthetic insulin is
developed by using bacteria as vectors and host cells.
b) Vaccine It is a biological substance prepared from the suspension of weak
or dead pathogenic (disease-causing) cells. It is introduced in the
body to enhance the production of antibodies (a protein in our
body that detects harmful substances) against particular antigens
(any substance that may cause harm to our body) such as rabies,
measles, flu, colds, Covid- 19 virus and others.
They are chemical substances used against bacterial infections.
c) Antibiotics They are produced by cultivating and manipulating fungal cells.

d) Production Some useful enzymes such as urokinase, which is used to dissolve

Of Enzymes blood clots, are also produced through rDNA technology
e) Solution Of When a man disputes that he is not the biological father of a child,
Disputed in contrary to what the mother has claimed, a DNA test will
Parentage confirm or disprove his claim.

B. Agriculture

a Transgenic Animals
- Animals which are engineered to carry genes from other organisms.

Example:

- Cow that modified to have human proteins in their milk.

- Transgenic fishes like salmon that contains human growth gene.

b Transgenic Plants
- Plants that are designed to be resistant to diseases, pests, herbicides, and droughts

Example:

Bt corn in the Philippines was engineered to be specifically resistant to the Asiatic Corn Borer (ACB), Ostrinia
furnacalis (Guenee), the most devastating corn pests in the industry.

The Bt eggplant contains a natural protein from the soil bacterium Bacillus thuringiensis which makes it resistant to
eggplant fruit and shoot borer (EFSB), the most destructive pest of eggplant.

Bt stands for Bacillus thuringiens - common soil in bacterium that contains a gene that produces a protein harmful
to fruit and shoot borer insects

C. Industry

The development of IMPORTANT CHEMICAL COMPOUNDS, IMPROVED FERMENTATION

PROCESS, AND PROTEIN FROM WASTE MATERIALS can be attained by designing and developing more
efficient varieties of microscopic organism. Effective and cheap biopharmaceutical products particularly, therapeutic
proteins to treat diseases in plants and animals are also great contribution of rDNA technology to industry.
Recombinant DNA methods are also employed as answer to environmental issues by converting wastes, and by
detecting arsenic and other contaminants in drinking water.