General Biology 2

Quarter 3 - Module 1
GENETICS

Prepared by: Ms. MARIA GLAIDYL P. FLORES

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 Module 1
Genetics

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.

This module has two (2) lessons:

 Lesson 1: Genetic Engineering
 Lesson 2: Applications of Recombinant DNA

Objective;

After going through this module, you are expected to:

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

2. Discuss the applications of recombinant DNA. (STEM_BIO11/12-IIIa-b-7)

3. Describe general features of the history of life on Earth, including generally

accepted dates and sequence of the geologic time scale and characteristics of
major groups of organisms present during these time periods. (STEM_BIO11/12-
IIIc-g-8)

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 Pre-Activity

Definition of Terms:

1. Genetic Engineering
2. DNA
3. Recombinant DNA
4. Plasmids
5. Cloning
6. Genome
7. Gene Mapping
8. Biotechnology
9. Polymerase Chain Reaction
10. Gene Therapy

1.How organisms may be modified?

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2. Enumerate plants and animals that have desirable or enhanced traits and how each of the
traits was introduced or developed. Modifying Technique ex. Classical Breeding, Recombinant
DNA Technology.

ENHANCED TRAIT MODIFYING TECHNIQUE

Example: Flavr-Savr (Delayed Ripening Recombinant DNA Technology
Tomatoes
1. 1.
2. 2.
3. 3.
4. 4.
5. 5.

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 Genetic Engineering

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.
https://www.britannica.com/science/genetic-engineering

 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.
https://www.sciencedaily.com/terms/plant_breeding.htm#:~:text=Classical%20plant%20breedi
ng%20uses%20deliberate,into%20a%20new%20genetic%20background.

 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.
https://www.genome.gov/genetics-glossary/Genetic
Engineering#:~:text=Genetic%20engineering%20is%20the%20process,selecting%20offspring%2
0with%20desired%20traits.

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; and
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

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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 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)

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 ACTIVTY 1

Performance Task:

Poster Making:

Create a poster on the steps and other methods involved in recombinant DNA.

POST QUIZ:

1. Determine which technologies are most appropriate for which cell types.

TECHNOLOGY CELL TYPE

1 Plants cells
2. Electroporation
3.Biolistics
4. Bacterial cells
5. Mammalian cells

1. Research on the pros and cons of genetic engineering.

PROS CONS
1.
2.
3.
4.
5.
2. What is your opinion on Genetic Engineering? Note: Support your opinion with facts and
include the issue of biosafety.

RECOMMENDED READINGS:

1. https://www.ck12.org/book/human-biology-genetics/section/10.1/
2.https://www.ck12.org/c/biology/biotechnology/lesson/Biotechnology-
BIO/?referrer=concept_details
3.https://www.khanacademy.org/science/biology/biotech-dna-technology/intro-to-biotech-
tutorial/a/intro-to-biotechnology

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 Pre-Activity

PRIOR KNOWLEDGE: Definition of Terms

1. Clone
2. Plasmids
3. Biotechnology
4. PCR Amplification
5. Detection
6. Modified Trait
7. Human Genome
8. Genetic Modified Organism

Designer Genes Work

1. How does DNA Replicate?

2. What is Genetically Modified Organism (GMO)?
3. Illustrate your own Designer genes based on the following:
1. Identify a special trait.
2. Identify a source organism.
3. Identify a target organism.
4. Identify the modified/ added trait.
Example: Hot Tomato > Chili > Tomato > Spicy Tomato

Tomatoes

It was reported this week that Brazilian scientists are hoping to create spicy tomatoes using
Crispr gene-editing techniques. Although tomatoes contain the genes for capsaicinoids (the
chemicals that give chillies their heat) they are dormant – Crispr could be used to make them
active. This is desirable because, compared to tomatoes, chillies are difficult to farm – and
capsaicinoids have other useful applications besides their flavour – in pepper spray for example.
https://www.theguardian.com/science/2019/jan/13/the-five-genetically-modified-fruit-edited-
bananas-tomatoes

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 Discuss the Applications of
Recombinant DNA

INTRODUCTION:

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 RECIPIENT APPLICATION (FIELD)

MODIFICATION ORGANISM
Insulin Production Insertion of Human Bacteria (Medicine)
Insulin Gene Production of HumanInsulin
in Bacteria
Pest Resistance Insertion of Bt-toxin Corn / (Agriculture)
gene Maize Production of cornplants
with increasedresistance to
cornboxer
Delayed Ripening Disruption of a gene Tomato Agriculture)
for a ripening plant Production of plantswith
enzyme fruits that havedelayed
(e.g. ripeningfruits. These fruits
polygalacturonase) willsurvive longertransport
time,allowing their
deliveryto further
locations(i.e. export
deliveries)
Chymosin Insertion of a gene Bacteria (Industry)
Production for Enhance large
chymosin scaleproduction ofchymosin.
This enzymeserves as a
substitutefor rennet in
thecoagulation of
milk.Rennet has to
beharvested from calves.The
large scaleproduction of
thisenzyme in
bacteriaprovides an
abundantsupply of
thisimportant componentfor
the cheeseproduction
industry.

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  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 (heat-resistant) 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;

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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)
Direction of elongation  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 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) ofa 43bp long gene, and the reverse primer binds at a position complementary to
nucleotide 39 of thecoding strand, then a 37bp product is expected per cycle of PCR.

PCR Applications

 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

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 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.

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 ACTIVTY 2

PERFORMANCE TASK

1. Illustrate the steps in restriction digestion and PCR.

POST QUIZ:

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.
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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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RECOMMENDED READINGS:

1.https://flexbooks.ck12.org/cbook/ck-12-middle-school-life-science
2.0/section/3.18/primary/lesson/recombinant-dna-ms-ls
2. https://www.ck12.org/book/cbse_biology_book_class_xii/section/14.1/
3. https://www.ck12.org/section/dna-technology/

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