Recombinant DNA Tech

WEEKLY LEARNING ACTIVITY SHEETS
Earth and Life Science Quarter 2 Week 4
RECOMBINANT DNA TECHNOLOGY
Learning Competency:
1. Describe the process of genetic engineering (S11/12LT-IIej-17)
1.1. Define recombinant DNA or genetic engineering;
1.2. Give a description of how recombinant DNA is created ;
1.3. Explain how recombinant DNA is and can be used.
Time Allotment: 120 minutes
Key Concepts
 Genetic engineering, also called recombinant DNA technology, involves the group of
techniques used to cut up and join together genetic material, especially DNA from different
biological species, and to introduce the resulting hybrid DNA into an organism in order to form
new combinations of heritable genetic material.
 Artificial selection is the most traditional form of genetic engineering, wherein
specificity of synthesis of target DNA sequence is less than current genetic engineering
technology. It has application on the pharmaceutical, industrial, agricultural, medical and
other industries.
Tools of Recombinant DNA Technology

The tools mainly include the following:
1. Enzymes
 restriction enzymes – help to cut
 polymerases- help to synthesize
 ligases- help to bind.
The restriction enzymes (help to cut) used in recombinant DNA technology play a
major role in determining the location at which the desired gene is inserted into the vector
genome. They are proteins that recognizes a specific, short nucleotide sequence and cuts
the DNA only at that specific site, which is known as restriction site or target sequence.
This gives rise to sticky ends in the sequence. The desired genes and the vectors are cut by
the same restriction enzymes to obtain the complementary sticky notes, thus making the
work of the ligases easy to bind the desired gene to the vector. Restriction enzymes are
found in bacteria (and other prokaryotes). More than 400 restriction enzymes have been
isolated from the bacteria that manufacture them. They are named for the organism from
which they were first isolated.
e.g.
EcoRI is isolated from E. coli strain RY13.
BamHI is isolated from Bacillus amyloliquefaciens strain H
Sau3A is isolated from Staphylococcus aureas strain 3A.
2 Types of Restriction Enzymes:
a. Endonucleases
b. Exonucleases.
Endonuclease is a group of enzymes that cleave (to divide or split) the phosphodiester
bond present within a polynucleotide chain. It cleaves the nucleotide sequence from the
Author: Iries Jane M. Concha-Calumbay

School/Station: Los Arcos National High School
Division: DepEd Agusan del Sur
email address: iriesjane.concha@deped.gov.ph
middle. It might form either sticky or blunt ends. Specific endonucleases, also called
restriction endonucleases cleave specific sites within a DNA sequence. They have
defensive properties against the entry of pathogenic microorganisms and can cleave specific
sites within a circular DNA. EcoRI, BamHI, Deoxyribonulcease I are some examples of
endonucleases. (Sapkota 2020)
Exonucleases are enzymes that cleave DNA sequences in a polynucleotide chain from
either the 5’ or 3’ end one at a time. It might form sticky ends. Exonuclease cleave in usually
non-specific sites and do not have defensive properties. They have less activity towards
circular DNA as compared to linear DNA. Snake venom, Exonuclease I, Xrn1 are some
examples of exonucleases. (Sapkota 2020)
Figure 1. The difference between endonuclease and exonuclease activity

Source:https://i0.wp.com/geneticeducation.co.in/wpcontent/uploa
ds/2020/07/Genetic-education-.001.jpeg?w=714&ssl=1
DNA polymerase (DNAP) (help to synthesize) is a type of enzyme that is responsible

for forming new copies of DNA, in the form of nucleic acid molecules. Nucleic acids are
polymers, which are large molecules made up of smaller, repeating units that are chemically
connected to one another. DNA is composed of repeating units called nucleotides or
nucleotide bases. DNA polymerase is responsible for the process of DNA replication,
during which a double-stranded DNA molecule is copied into two identical DNA molecules.
Scientists have taken advantage of the power of DNA polymerase molecules to copy DNA
molecules in test tubes via polymerase chain reaction, also known as PCR.
DNA ligase (help to bind) is a DNA-joining enzyme. It is a specific type of enzyme that
facilitates the joining of DNA strands together by catalyzing the formation of a
phosphodiester bond. It plays a role in repairing single-strand breaks in duplex DNA in
living organisms, but some forms (such as DNA ligase IV) may specifically repair double-
strand breaks (i.e. a break in both complementary strands of DNA). Single-strand breaks
are repaired by DNA ligase using the complementary strand of the double helix as a
template, with DNA ligase creating the final phosphodiester bond to fully repair the DNA.
DNA ligase is used in both DNA repair and DNA replication. Purified DNA ligase is used in
gene cloning to join DNA molecules together to form recombinant DNA.

2. Vectors
It help in carrying and integrating the desired gene. These form a very important part of
the tools of recombinant DNA technology as they are the ultimate vehicles that carry forward
the desired gene into the host organism.
Most common vectors in rDNA technology
Plasmids and bacteriophages are the most common vectors in recombinant DNA
technology that are used as they have very high copy number. The vectors are made up of
an origin of replication- This is a sequence of nucleotide from where the replication starts,
a selectable marker – constitute genes which show resistance to certain antibiotics like
ampicillin; and cloning sites – the sites recognized by the restriction enzymes where desired
DNAs are inserted.
3. Host Organism
It is into which the recombinant DNA is introduced. The host is the ultimate tool of
recombinant DNA technology which takes in the vector engineered with the desired DNA
with the help of the enzymes. There are a number of ways in which these recombinant DNAs
are inserted into the host, namely – microinjection, biolistic or gene gun, alternate
cooling and heating, use of calcium ions, etc.
Steps in Genetic Engineering or Recombinant DNA Technology
1. Selection and Isolation Of DNA Insert

First step in recombinant DNA technology (rDNA) is the selection of a DNA segment
of interest which is to be cloned. This desired DNA segment is then isolated enzymatically.
This DNA segment of interest is termed as DNA insert or foreign DNA or target DNA or
cloned DNA.
2. Selection of Suitable Cloning Vector
A cloning vector is a self-replicating DNA molecule, into which the DNA insert is to
be integrated. A suitable cloning vector is selected in the next step of rDNA technology.
Most commonly used vectors are plasmids and bacteriophages.
3. Introduction of DNA-Insert into Vector to Form rDNA Molecule
The target DNA or the DNA insert which has been extracted and cleaved
enzymatically by the selective restriction endonuclease enzymes in step 1 are now ligated
(joined) by the enzyme ligase to vector DNA to form rDNA molecule which is often called
as cloning-vector-insert DNA construct.
4. Recombinant DNA Molecule is introduced into a Suitable Host
Suitable host cells are selected and the rDNA molecule so formed step 3 is introduced
into these host cells. This process of entry of rDNA into the host cell is called
transformation. Usually selected hosts are bacterial cells like E. coli, however yeast
and fungi may also be utilized.
5. Selection of Transformed Host Cells
Transformed cells (or recombinant cells) are those host cells which have taken up
the rDNA molecule. In this step the transformed cells are separated from the non-
transformed cells by using various methods making use of marker genes.
6. Expression and Multiplication of DNA Insert in the Host
Finally, it is to be ensured that the foreign DNA inserted into the vector DNA is
expressing the desired character in the host cells. Also, the transformed host cells are
multiplied to obtain sufficient number of copies. If needed, such genes may also be
transferred and expressed into another organism.

Figure 2. The basic technique for creating a genetically engineered bacterial cell
Source:https://bodell.mtchs.org/OnlineBio/BIOCD/text/chapter
13/concept13.2.html
Figure 3. The splicing together fragments of DNA from two different sources to produce a
recombinant DNA molecule.
Source:https://bodell.mtchs.org/OnlineBio/BIOCD/text/chapter
13/concept13.2.html

Figure 4. The simplified process of recombinant DNA technology using the bacterial plasmid
as cloning vector
Source:https://bodell.mtchs.org/OnlineBio/BIOCD/text/chapter13/concept13.2.html

Application of Recombinant DNA Technology
1. Production of Transgenic Plants

By utilizing the tools and techniques of genetic engineering it is possible to produce
transgenic plants or the genetically modified plants. Many transgenic plants have been
developed with better qualities like resistance to herbicides, insects or viruses or with
expression of male sterility etc.
2. Production of Transgenic Animal
By the use of rDNA technology, desired genes can be inserted into the animal so as
to produce the transgenic animal. The method of rDNA technology aids the animal breeders
to increase the speed and range of selective breeding in case of animals. It helps for the
production of better farm animals so as to ensure more commercial benefits. Another
commercially important use of transgenic animals is the production of certain proteins and
pharmaceutical compounds. Transgenic animals also contribute for studying the gene
functions in different animal species. Biotechnologists have successfully produced
transgenic pigs, sheep, rats and cattle.
3. Production of Hormones
By the advent of techniques of rDNA technology, bacterial cells like E.coli are utilized
for the production of different fine chemicals like insulin, somatostatin, somatotropin and
endorphin.
Human Insulin Hormone i.e., Humulin is the first therapeutic product which was
produced by the application of rDNA technology.
Insulin is a natural hormone created by the pancreas that controls the amount of
glucose in the bloodstream at any given moment. It also helps store glucose in your liver,
fat, and muscles. Finally, it regulates the body’s metabolism of carbohydrates, fats, and
proteins. Without proper insulin function, the body can’t store glucose in the muscles or
liver, but neither can it make any fat. Instead, the fat breaks down and produces, among
other things, keto acids. Decreased or absent insulin activity results in diabetes mellitus, a
condition of high blood sugar level (hyperglycemia).
Human insulin is used to control blood sugar in people who have type 1 diabetes
(condition in which the body does not make insulin and therefore cannot control the amount
of sugar in the blood) or in people who have type 2 diabetes (condition in which the blood
sugar is too high because the body does not produce or use insulin normally) that cannot
be controlled with oral medications alone.
4. Biosynthesis of Interferon
Interferons are the glycoproteins which are produced in very minute amounts by
the virus-infected cells. Interferons have antiviral and even anti-cancerous properties. By
rDNA technology method, the gene of human fibroblasts (which produce interferons in
human beings) is inserted into the bacterial plasmid. These genetically engineered bacteria
are cloned and cultured so that the gene is expressed and the interferons are produced in
fairly high quantities. This interferon, so produced, is then extracted and purified.
5. Production of Commercially Important Chemicals
Various commercially important chemicals can be produced more efficiently by
utilizing the methods of rDNA technology. A few of them are the alcohols and alcoholic
beverages obtained through fermentation; organic acids like citric acid, acetic acid, etc.
and vitamins produced by microorganisms.
6. Production of Vaccines
Vaccines are the chemical preparations containing a pathogen in attenuated (or
weakened) or inactive state that may be given to human beings or animals to confer
immunity to infection. A number of vaccines have been synthesized biologically through
rDNA technology, these vaccines are effective against numerous serious diseases caused by
bacteria, viruses or protozoa. These include vaccines for polio, malaria, cholera, hepatitis,
rabies, smallpox, etc. The generation of DNA vaccines has revolutionized the approach of

treatment of infectious diseases. DNA-vaccine is the preparation that contains a gene
encoding an immunogenic protein from the concerned pathogen.
Table 1. Standard routine immunization schedule for infants in the Philippines
Source: https://www.doh.gov.ph/expanded-program-on-immunization
Table 2. Tetanus Toxoid Immunization Schedule for Women
Source: https://www.doh.gov.ph/expanded-program-on-immunization
7. Production of Antibiotics
Antibiotics produced by microorganisms are very effective against different viral,
bacterial or protozoan diseases. Some important antibiotics are tetracycline, penicillin,
streptomycin, novobiocin, bacitracin, etc. rDNA technology helps in increasing the
production of antibiotics by improving the microbial strains through modification of genetic
characteristics.

8. Application in Enzyme Engineering
As we know that the enzymes are encoded by genes, so if there are changes in a gene
then definitely the enzyme structure also changes. Enzyme engineering utilizes the same
fact and can be explained as the modification of an enzyme structure by inducing alterations
in the genes which encode for that particular enzyme.
Table 3. Food and Feed Enzymes from UP Los Baῆos National Institute of Molecular
Biology and Biotechnology
Name of Enzyme Usage
ALPHA-AMYLASE It is used for high glucose/fructose syrup and maltodextrin
production, improve dough quality and as feed additive.
CELLULASE It is used in ethanol production, essential oil extraction and as
feed additive for livestock and poultry.
GLUCOAMYLASE It is used to convert starchy materials such as cassava, corn,
and sweet potato into maltodextrin and glucose syrup.
LIPASE It is used to modify coconut oil for higher value beta-
monoglyceride.
PECTINASE It is used for injuice and wine clarification and essential oil
extraction.
PROTEASE Neutral - used to improve bread quality
Acid - as feed additive to improve growth performance of swine
and poultry
Alkaline - an additive for detergent
MICROBIAL RENNET It is used as a good substitute to animal rennet in cheese
making. The technology utilized local fungal strain called
Rhizopus chinensis.
XYNALASE It is used as animal feed additives and for improved cleaning
ability of detergent.
Source: https://biotech.uplb.edu.ph/products/food-and-feed-enzymes
9. Prevention and Diagnosis of Diseases

Genetic engineering methods and techniques have greatly solved the problem of
conventional methods for diagnosis of diseases. It also provides methods for the prevention
of a number of diseases like AIDS, cholera, etc. Monoclonal antibodies are useful tools for
disease diagnosis. Monoclonal antibodies are produced by using the technique called
hybridoma technology.
10. Gene Therapy
Gene therapy is undoubtedly the most beneficial area of genetic engineering for
human beings. It involves delivery of specific genes into human body to correct the diseases.
Thus, it is the treatment of diseases by transfer and expression of a gene into the patients’
cells so as to ensure the restoration of a normal cellular activity.
11. Practical Applications of Genetic Engineering
Recombinant DNA technology has an immense scope in Research and Experimental
studies. It is applied for: a. Localizing specific genes. b. Sequencing of DNA or genes. c.
Study of mechanism of gene regulation. d. Molecular analysis of various diseases. e. Study
of mutations in DNA, etc.
12. Applications in forensic science
The applications of rDNA technology in forensic sciences largely depend on the
technique called DNA profiling or DNA fingerprinting. It enables us to identify any person
by analyzing his hair roots, wood stains, serum, etc. DNA fingerprinting also helps to solve
the problems of parentage and to identify the criminals.
13. Biofuel Production
Biofuels are derived from biomass and these are renewable and cost effective.
Genetic engineering plays an essentially important role in a beneficial and largescale

production of biofuels like biogas, bio hydrogen, biodiesel, bio-ethanol., etc. Genetic
engineering helps to improve organisms for obtaining higher product yields and product
tolerance.
14. Genetically stable high producing microorganisms
Genetically stable high producing microorganisms are being developed by using
modern rDNA techniques, which aid in an efficient production of bioenergy.
15. Energy crop plants
The energy crop plants are those plants which use solar energy in a better way for
production of biomass. Genetic improvements of these energy crop plants greatly help for
quick and high production of biomass which in turn reduces the biofuel production cost.
The fermenting microbes which are utilized for biogas production are improved at the
genetic level for achieving better result.
16. Environment Protection
Genetic engineering makes its contributions to the environment protection in various
ways. Most important to mention are the new approaches utilized for waste treatments and
bioremediation. Environment protection means the conservation of resources and hence to
limit the degradation of environment.
Exercises / Activities
Activity 1: At the Right Place, At the Right Time
Reference: https://www.tes.com/teaching-resource/what-is-genetic-engineering-11149041
Objective: 1. Determine the correct sequence of how human insulin is produced as a process of
genetic engineering
What you need: pen and paper
What to do:
1. Sort the following statements into the correct order to describe how human insulin is
produced.
2. In a one whole sheet of paper, copy the statement that comes first and put number “1”
before the statement followed by the second step, then third and so on.
___________The insulin can be removed and purified

and used by people with diabetes.
___________DNA is taken from a human cell.
___________The plasmid with the insulin gene is

inserted back into the bacteria.
___________The same restriction enzyme is used to

cut a section of DNA from the plasmid.
___________A restriction enzyme is used to cut out

the gene for insulin from the DNA.
___________The bacteria are left to divide and

reproduce.
___________An enzyme is then used to cut the

plasmid (DNA) ring out of a bacterial cell.
Figure 5. Human insulin production

Source: https://www.ck12.org/book/human-biology-
genetics/section/10.1/

Reflection
Write 5-sentence answer in a separate sheet of paper analyzing the importance of genetic
engineering or rDNA Technology in vaccine development and QR code development for contact
tracing to fight Covid-19.
Rubrics:
Score Indicators
Practical application is scientifically explained consistent to the concepts, and has
15
no misconception.
Practical application is scientifically explained consistent to the concepts, but with
10
minimal misconception.
Practical application is explained consistent to the concepts but with
5
misconceptions.
0 No discussion.
References:
Sakpota, Anupama. “11 differences between Endonuclease and Exonuclease”. July 20, 2020
S. A. Shinde et al /Int.J. MediPharm Res. 2018, 4(2), pp 79-88. 84 3.
https://biotech.uplb.edu.ph/
The Commision on Higher Educationin collaboration with Philippine Normal University. “Lesson
33: Perpetuation of Life.” Earth and Life Science: Teaching Guide for Senior High School, Quezon
City, Commission on higher Education, 2016, pp 215-218.
Photos
“Figure 1”. Io.wp.com. Accessed November 16, 2020

https://i0.wp.com/geneticeducation.co.in/wpcontent/uploads/2020/07/Genetic-education-
.001.jpeg?w=714&ssl=1
“Figure 2”. Bodell.mtchs.org. Accessed October 28, 2020

https://bodell.mtchs.org/OnlineBio/BIOCD/text/chapter13/concept13.2.html


“Table 1”. Doh.gov.ph. Accessed November 16, 2020

https://www.doh.gov.ph/expanded-program-on-immunization

Activity 1: AT THE RIGHT PLACE, AT THE RIGHT TIME
1. DNA is taken from a human cell.
2. A restriction enzyme is used to cut out the gene for insulin from the DNA.
3. The same restriction enzyme is used to cut a section of DNA from the plasmid.
4. An enzyme is then used to cut the plasmid (DNA) ring out of a bacterial cell.
5. The plasmid with the insulin gene is inserted back into the bacteria.
6. The bacteria are left to divide and reproduce.
7. The insulin can be removed and purified and used by people with diabetes.
Answer Key
https://www.ck12.org/book/human-biology-genetics/section/10.1/
“Figure 5”. Accessed November 5, 2020
https://www.doh.gov.ph/expanded-program-on-immunization
“Table 2”. Doh.gov.ph. Accessed November 16, 2020

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WEEKLY LEARNING ACTIVITY SHEETS

Earth and Life Science Quarter 2 Week 4

RECOMBINANT DNA TECHNOLOGY

Tools of Recombinant DNA Technology

Author: Iries Jane M. Concha-Calumbay

Figure 1. The difference between endonuclease and exonuclease activity

DNA polymerase (DNAP) (help to synthesize) is a type of enzyme that is responsible

Author: Iries Jane M. Concha-Calumbay

Steps in Genetic Engineering or Recombinant DNA Technology

1. Selection and Isolation Of DNA Insert

Author: Iries Jane M. Concha-Calumbay

Author: Iries Jane M. Concha-Calumbay

Author: Iries Jane M. Concha-Calumbay

1. Production of Transgenic Plants

Author: Iries Jane M. Concha-Calumbay

Table 1. Standard routine immunization schedule for infants in the Philippines

Table 2. Tetanus Toxoid Immunization Schedule for Women

Author: Iries Jane M. Concha-Calumbay

9. Prevention and Diagnosis of Diseases

Author: Iries Jane M. Concha-Calumbay

___________The insulin can be removed and purified

___________DNA is taken from a human cell.

___________The plasmid with the insulin gene is

___________The same restriction enzyme is used to

___________A restriction enzyme is used to cut out

___________The bacteria are left to divide and

___________An enzyme is then used to cut the

Figure 5. Human insulin production

Author: Iries Jane M. Concha-Calumbay

“Figure 1”. Io.wp.com. Accessed November 16, 2020

“Figure 2”. Bodell.mtchs.org. Accessed October 28, 2020

“Figure 3”. Bodell.mtchs.org. Accessed October 28, 2020

“Figure 4”. Bodell.mtchs.org. Accessed October 28, 2020

“Table 1”. Doh.gov.ph. Accessed November 16, 2020

Author: Iries Jane M. Concha-Calumbay

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