This module demonstrates your understanding of the characteristics of Earth that are necessary to support life,
particularly on the essential components of this planet that drives all living things or biotic factors (plants, animals,
microorganisms) to exist. It also emphasizes on the different subsystems (geosphere, hydrosphere, atmosphere, and biosphere)
that make up the Earth and how these systems interact to produce the kind of Earth we live in today.
This module will help you explore the key concepts on topics that will help you answer the questions pertaining to our very
own, planet earth.
Learning Competency
The learners should be able to outline the steps involved in genetic engineering.
Lesson 1 Genetic Engineering
What’s is it
INTRODUCTION:
❖ Genetic engineering, the artificial manipulation, modification, and recombination of DNA or other nucleic acid molecules
in order to modify an organism or population of organisms.
❖ The term genetic engineering initially referred to various techniques used for the modification or manipulation of organisms
through the processes of heredity and reproduction. As such, the term embraced both artificial selection and all the
interventions of biomedical techniques, among them artificial insemination, in vitro fertilization (e.g., “test-tube” babies),
cloning, and gene manipulation.
❖ Classical plant breeding uses deliberate interbreeding (crossing) of closely or distantly related individuals to produce new
crop varieties or lines with desirable properties. Plants are crossbred to introduce traits/genes from one variety or line into a
new genetic background.
❖ 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 the direct manipulation of one or more genes. Most often, a gene from another
species is added to an organism's genome to give it a desired phenotype.
Genetic engineering involves the use of molecular techniques to modify the traits of a target organism. The
modification of traits may involve:
1. introduction of new traits into an organism
2. enhancement of a present trait by increasing the expression of the desired gene
3. enhancement of a present trait by disrupting the inhibition of the desired genes’ expression.
A general outline of recombinant DNA may be given as follows:
1. cutting or cleavage of DNA by restriction enzymes (REs)
2. selection of an appropriate vector or vehicle which would propagate the recombinant DNA ( eg. circular plasmid
in bacteria with a foreign gene of interest)
3. ligation (join together) of the gene of interest (eg. from animal) with the vector (cut bacterial plasmid)
4. transfer of the recombinant plasmid into a host cell (that would carry out replication to make huge copies of the
recombined plasmid)
5. selection process to screen which cells actually contain the gene of interest
6. sequencing of the gene to find out the primary structure of the protein
Ways in which these plasmids may be introduced into host organisms:
❖ Biolistics. In this technique, a “gene gun” is used to fire DNA-coated pellets on plant tissues. Cells that survive the
bombardment, and are able to take up the expression plasmid coated pellets and acquire the ability to express the designed
protein.
❖ Plasmid insertion by Heat Shock Treatment. Heat Shock Treatment is a process used to transfer plasmid DNA into bacteria.
The target cells are pre-treated before the procedure to increase the pore sizes of their plasma membranes. This pretreatment
(usually with CaCl2) is said to make the cells “competent” for accepting the plasmid DNA. After the cells are made competent,
they are incubated with the desired plasmid at about 4°C for about 30min. The plasmids concentrate near the cells during this
time. Afterwards, 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 is believed to increase and decrease the pore sizes in the membrane. The
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plasmid DNA near the membrane surface are taken into the cells by this process. The cells that took up the plasmids acquire
new traits and are said to be “transformed”.
❖ Electroporation. This technique follows a similar methodology as Heat Shock Treatment, but, the expansion of the
membrane pores is done through an electric “shock”. This method is commonly used for insertion of genes into mammalian
cells.
Some methods are:
• Selection of plasmid DNA containing cells
• Selection of transformed cells with the desired gene
• PCR detection of plasmid DNA
• Genetically Modified Organisms (GMOs)
EVALUATION
1. Research on the pros and cons of genetic engineering.
PROS
CONS
1.
1.
2.
2.
3.
3.
4.
4.
5.
5.
2. What is your opinion on Genetic Engineering? Note: Support your opinion with facts and include the issue of biosafety.
Lesson 2 Discuss the Applications of Recombinant DNA
PRESENTATION OF RECOMBINANT DNA
There are many different traits that can be introduced to organisms to change their properties. The following table shows
examples of modified traits using cloned genes and their applications:
MODIFIED TRAIT
GENE MODIFICATION
Insulin Production
Insertion of Human
Insulin Gene
RECIPIENT
ORGANISM
Bacteria
Pest Resistance
Insertion of Bt-toxin gene
Corn / Maize
Delayed Ripening
Disruption of a gene
for a ripening enzyme
(e.g.
polygalacturonase)
Tomato plant
Chymosin Production
Insertion of a gene for
chymosin
Bacteria
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APPLICATION
(FIELD)
(Medicine)
Production of Human
Insulin in Bacteria
(Agriculture)
Production of corn
plants with increased
resistance to corn
boxer
Agriculture)
Production of plants
with fruits that have
delayed ripening
fruits. These fruits will
survive longer
transport time,
allowing their delivery
to further locations
(i.e. export deliveries)
(Industry)
Enhance large scale
production of
chymosin. This enzyme
serves as a substitute
for rennet in the
coagulation of milk.
Rennet has to be
harvested from calves.
The large scale
production of this
enzyme in bacteria
provides an abundant
supply of this
important component
for the cheese
production industry.
PCR Amplification
Once a desired trait is chosen, information must be acquired for either its detection or expression in a given organism.
1. Detection
❖ Some researchers may be interested in determining if a given gene/trait is available in a particular organism. If no
previous research provides this information, researchers may test the DNA of different organisms for the presence of these
specific genes. A technique that allows the detection of specific genes in target organisms is called PCR.
❖ PCR amplification is an in-vitro method that simulates DNA replication in vivo. It utilizes a thermostable (heatresistant) DNA polymerase that builds single stranded DNA strands unto unwound DNA templates.
❖ PCR uses repeated cycles of incubation at different temperatures to promote the unwinding of the DNA template
(~95°C); the annealing of a primer (a ~20bp oligonucleotide sequence (recall RNA primers in DNA replication) onto the
ssDNA template strand (~54 - 60°C); and the extension of the generated ssDNA strand through the binding of complementary
bases to the template strand (~72° C). The thermostability of the polymerase allows it to survive the repeated cycles of
denaturation, annealing and extension with little loss of enzyme function. Each cycle of PCR doubles the amount of the
target sequence. A typical PCR experiment uses about 35 cycles of amplification. This increases the original amount of the
target sequence by 235 (i.e. ~34 billion) times.
❖ Gene detection by PCR involves the design of primers that would only bind to sequences that are specific to a target.
For example, researchers would want to find out if gene X (e.g. the gene for insulin) is available in a target organism (e.g. a
mouse, Mus musculus). Primers may be designed by looking at the available sequences for gene X in the databases (e.g. all
the genes for insulin in different organisms; humans, pigs, cows, etc.). The different gene X sequences must be aligned/
compared to match areas of sequence similarity (conserved sequences) and areas of sequence dissimilarity (non-conserved
sequences). Primers designed to have the same sequence as the conserved areas will be specific for binding gene X sequences
in all the target organisms. Primers designed to have the same sequence as the non-conserved areas will only be specific for
the organisms which match its sequence.
STEPS in PCR Amplification
Step 0: Undenatured Template ; Temp ~ 54 °"C;
Template: double stranded (ds) DNA strand. Complementary sequences are held together by H-bonds
5’ A T GCGATGAGGATATGACCCGATAGATAGAGGTATCTAGAGAT 3’ (Coding strand)
3’ T A CGCTACTCCTATACTGGGCTATCTATCTCCATAGATCTCTA 5’ (Non-coding strand)
Step 1: Template denaturation ; Temp ~ 95 °"C;
Template: single stranded (ss) DNA strands; DNA strands are separated; H-bonds between complementary sequences are
broken
5’ A T GCGATGAGGATATGACCCGATAGATAGAGGTATCTAGAGAT 3’ (Coding strand)
3’ T A CGCTACTCCTATACTGGGCTATCTATCTCCATAGATCTCTA 5’ (Non-coding strand)
Step 2: Primer Annealing ; Temp ~ 54 °"C (dependent on primer melting temperature);
Template: ssDNA strands. H-bonds are formed between complementary sequences on the primers and the target sequences.
5’ A T GCGATGAGGATATGACCCGATAGATAGAGGTATCTAGAGAT 3’ (Coding strand)
CCATAGATC (Reverse Primer)
5’ GCGATGAGG 3’ Direction of elongation (Forward Primer)
3’ T A CGCTACTCCTATACTGGGCTATCTATCTCCATAGATCTCTA 5’ (Non-coding strand)
Step 3: New DNA strand elongation ; Temp ~ 72 °"C;
The two new dsDNA strands are formed by the elongation of the generated ssDNA and the H-bonds between the
complementary sequences on these new strands and their templates. Each of the new dsDNA strands is made up of one old
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strand from the original template, and one new strand that was generated as a reverse complement of the template. This is
called semiconservative replication of the sequence.
New Strand 1:
5’ A T GCGATGAGGATATGACCCGATAGATAGAGGTATCTAGAGAT 3’ (Coding strand) (old)
3’ CGCTACTCCTATACTGGGCTATCTATCTCCATAGATC-5’ (Reverse Primer) (new)
New Strand 2:
5’ GCGATGAGGATATGACCCGATAGATAGAGGTATCTAG-3’ (Forward Primer) (new)
3’ T A CGCTACTCCTATACTGGGCTATCTATCTCCATAGATCTCTA 5’ (Non-coding strand) (old)
Step 4: Repeat step 1 to 3 for N number of cycles (N is usually 35)
PCR Results
The expected product of PCR amplification will depend on the sequences / position at which the primer sequences
bind. If the forward primer starts binding at nucleotide 3 (coming from the 5’ end) of a 43bp long gene, and the reverse primer
binds at a position complementary to nucleotide 39 of the coding strand, then a 37bp product is expected per cycle of PCR.
PCR Applications
1. PCR may be used to detect the presence of a desired gene in an organism. Depending on the primer design, the
expected product may represent only a specific region of the gene or the entire gene itself. The first case is useful for
detection of the gene, or the detection of organisms with that specific gene within a sample. The second case is useful
for the amplification of the entire gene for eventual expression in other organisms. The direct amplification/copying
of a full gene is part of the process for “cloning” that gene.
2. Cloning and Expression. Some genes provide economically, and industrially important products (e.g. insulin-coding
genes; genes for collagen degradation). In some cases, scientists would want to put these genes into organisms for the
expression of their products. One example would be the insertion of an insulin- coding gene from the human genome
into bacteria. This allows the “transformed” bacteria to now produce human insulin as a product. Certain types of
bacteria are capable of this process since they are able to take genes within their cell membranes for eventual
expression. The genes are normally in the form of small, circular DNA structures called plasmids.
EVALUATION
1. Discuss how PCR may be used for the detection of disease-causing pathogens in a population during the COVID Pandemic.
For example: it may be used to check if a patient has a COVID virus infection.
2. Discuss how the cloning and expression of certain genes allows for massive production of the desired product.
For Example: the cloning and expression of insulin in bacteria allows for the mass production of this necessary protein for
use by diabetic patients.
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