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Biotechnology
Quarter 3 – Module 3:
Steps in Recombinant
DNA
Technology
 What I Need to Know

This module was designed and written with you in mind. It is here to help
you master the steps in Recombinant DNA Technology. The scope of this module
permits it to be used in many different learning situations. The language used
recognizes the diverse vocabulary level of students. The lessons are arranged to
follow the standard sequence of the course. But the order in which you read
them can be changed to correspond with the textbook you are now using.

The module is about:

● Steps in Recombinant DNA Technology

After going through this module, you are expected to:

1. Outline the steps in Recombinant DNA Technology

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 What I Know

Directions: Read each question carefully. Choose the letter of the best answer.

1. Which refers to the combination of two DNA strands that are constructed
artificially?
a. Genetic Material
b. Recombinant Cells
c. Recombinant DNA
d. Restriction Enzymes

2. Which among the following is the Blueprint of Life?

a. Cell
b. DNA
c. Protein
d. RNA

3. Which refers to the small accessory ring of the DNA in some bacteria? a.
Interferon
b. Plasmid
c. Restriction enzymes
d. Vector

4. What molecule is being used to cut a specific area of the DNA?

a. Agarose Gel
b. Plasmid
c. Recombinant Cells
d. Restriction enzymes

5. An organism that contains genetic material from two different organisms

is called ___________. a. Clone
b. GMO
c. Mutant
d. Restriction enzymes

6. What technique is being used to make a million copies of a sample DNA?

a. DNA Fingerprinting
b. Gel Electrophoresis
c. Gene Therapy
d. Polymerase Chain Reaction

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7. Which among the following does NOT describe the principle of Gel
Electrophoresis?
a. DNA fragments are separated on the basis of size.
b. The DNA fragments will move towards the negative charge.
c. The smallest DNA fragments will move faster than the larger DNA
fragments.
d. DNA fragments are injected into wells and an electric current is applied
along with the gel.

8. What equipment is used to amplify the DNA?

a. Centrifuge Machine
b. Microscope
c. Pipette
d. Thermal Cycler

9. Which among the following serves as a starting point for DNA synthesis?
a. DNA polymerase
b. Primer
c. Restriction enzymes
d. Taq polymerase

10. What organism is being used to transfer foreign genetic material into a
cell? a. DNA
b. Plasmid
c. Restriction enzymes
d. Vector

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 Lesso
Steps in Recombinant
n 1 DNA Technology
What’s In

Activity 1
Direction: Write True if the statement is correct and False if incorrect.

1. Genetic Engineering is the artificial manipulation of DNA or other nucleic

acid molecules in order to modify an organism or
population of organisms.
2. Restriction enzyme was discovered by Werner Arber.
3. Genes are small rings of DNA.
4. DNA technology makes it possible to clone genes for basic
research and commercial applications.
5. Restriction enzymes cut the DNA into a particular site.
In our previous lesson, we learned how genetic materials are manipulated.
One of the ways is through Genetic engineering. In Genetic engineering, genes
are manipulated for practical purposes.

Genetic Engineering

DNA technology has launched a revolution in Biotechnology. DNA technology

(via gene manipulation) makes it possible to clone genes for basic research and
commercial applications. DNA technology is applied to areas ranging from
agriculture to criminal law. One example of DNA technology is Genetic
engineering. Genetic Engineering is the the artificial manipulation, modification,
and recombination of DNA or other nucleic acid molecules in order to modify an
organism or population of organisms. In the latter part of the 20th century,
however, the term came to refer more specifically to methods of recombinant
DNA technology, in which DNA molecules from two or more sources are
combined either within cells or in vitro and are then inserted into host organisms
in which they are able to propagate.

The possibility for recombinant DNA technology emerged with the discovery
of restriction enzymes in 1968 by Swiss microbiologist Werner Arber.

Most recombinant DNA technology involves the insertion of foreign genes into
the plasmids of common laboratory strains of bacteria. Plasmids are small rings
of DNA; they are not part of the bacterium’s chromosome. Nonetheless, they are
capable of directing protein synthesis, and, like chromosomal DNA, they are
reproduced and passed on to the bacterium’s progeny. Thus, by incorporating
foreign DNA (for example, a mammalian gene) into a bacterium, researchers can
obtain an almost limitless number of copies of the inserted gene. Furthermore, if
the inserted gene is operative (if it directs protein synthesis), the modified

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bacteria will produce the protein specified by the foreign DNA. Editors of
Encyclopedia Britannica (2020).

What’s New

Activity 1. Steps in Recombinant DNA Technology

Directions: Arrange the steps in Recombinant DNA Technology in chronological
order. Use numbers 1-7.
A. Amplification Using PCR
B. Isolation of Recombinant Cell
C. Isolation of Genetic Material
D. Obtaining or culturing the Foreign Gene product.
E. Ligation of DNA Molecules.
F. Restriction Enzymes Digestion
G. Insertion of Recombinant DNA into Host

What is It

In this lesson, we shall outline the main steps in Recombinant DNA

Technology and the importance of Recombinant DNA Technology .

Recombinant DNA Technology

Recombinant DNA technology refers to the joining together of DNA

molecules from two different species that are inserted into a host organism to
produce new genetic combinations that are of value to science, medicine,
agriculture, and industry. Recombinant DNA (rDNA), on the other hand, is the
general name for a piece of DNA that has been created by the combination of at
least two different DNA strands. They are DNA molecules formed by laboratory
methods of genetic recombination to bring together genetic material from
multiple sources, creating sequences that would not otherwise be found in the
genome. Aryal (2018).

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Steps of Genetic Recombination Technology

1. Isolation of Genetic Material - Since DNA exists within the cell membrane
along with other macromolecules such as RNA, polysaccharides, proteins,
and lipids, it must be separated and purified which involves enzymes such as
restriction enzymes.

2. Restriction Enzymes Digestion - The technique ‘Agarose Gel

Electrophoresis’ reveals the progress of the restriction enzyme digestion.
This technique involves running out the DNA on an agarose gel. On the
application of current, the negatively charged DNA travels to the positive
electrode and is separated out based on size. This allows separating and
cutting out the digested DNA fragments. The vector DNA is also processed
using the same procedure.

3. Amplification Using PCR - Polymerase Chain Reaction or PCR is a method

of making multiple copies of a DNA sequence using the enzyme – DNA
polymerase in vitro. It helps to amplify a single copy or a few copies of DNA
into thousands to millions of copies. PCR reactions are run on thermal cyclers
using the following components: 1.)Template – DNA to be amplified
2.)Primers – small, chemically synthesized oligonucleotides that are
complementary to a region of the DNA. 3.) Enzyme – DNA polymerase
4.)Nucleotides – needed to extend the primers by the enzyme. 5.) The cut
fragments of DNA can be amplified using PCR and then ligated with the cut
vector.

4. Ligation of DNA Molecules – The purified DNA and the vector of interest
are cut with the same restriction enzyme. This gives us the cut fragment of
DNA and the cut vector that is now open. The process of joining these two
pieces together using the enzyme DNA ligase is ligation. The resulting DNA
molecule is a hybrid of two DNA molecules – the interest molecule and the
vector. In the terminology of genetics this intermixing of different DNA
strands is called recombination. Hence, this new hybrid DNA molecule is also
called a recombinant DNA molecule and the technology is referred to as the
recombinant DNA technology.

5. Insertion of Recombinant DNA into Host - In this step, the recombinant

DNA is introduced into a recipient host cell mostly, a bacterial cell. This
process is called transformation. Bacterial cells do not accept foreign DNA
easily. Therefore, they are treated to make them competent to accept new
DNA. The processes used may be thermal shock, Ca ++ ion treatment, and
electroporation.

6. Isolation of Recombinant Cells-The transformation process generates a

mixed population of transformed and non-transformed host cells. The
selection process involves filtering the transformed host cells only. For
isolation of recombinant cells from non-recombinant cells, a marker gene of
the plasmid vector is employed.

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7. Obtaining or culturing the Foreign Gene product - When you insert a
piece of alien DNA into a cloning vector and transfer it into a bacterial cell,
the alien DNA gets multiplied. The ultimate aim is to produce a desirable
protein expression. The cells harboring cloned genes of interest are grown on
a small scale in the laboratory. These cell cultures are used for extracting the
desired protein using various separation techniques.

Recombinant Human Growth Hormone

Source: https://simplebiologyy.blogspot.com/2016/02/process-of-recombinantdna-
technology-genetic-engineering.html

Importance of Recombinant DNA Technology

Recombinant DNA technology is playing a vital role in improving health

conditions by developing new vaccines and pharmaceuticals. The treatment
strategies are also improved by developing diagnostic kits, monitoring devices,
and new therapeutic approaches. Synthesis of synthetic human insulin and
erythropoietin by genetically modified bacteria and the production of new types
of experimental mutant mice for research purposes are some one of the leading
examples of genetic engineering in health. Likewise, genetic engineering
strategies have been employed to tackle environmental issues such as
converting wastes into biofuels and bioethanol, cleaning the oil spills, carbon,
and other toxic wastes, and detecting arsenic and other contaminants in drinking
water. The genetically modified microbes are also effectively used in biomining
and bioremediation.

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 What’s More

Activity 1. Enzymes in Recombinant DNA Technology

Directions: Draw a Venn Diagram to differentiate DNA Polymerase with DNA
Ligase.

DNA Polymerase DNA Ligase

Guide Questions
1. What is the first step in Recombinant DNA Technology?
2. In what step does the cut fragment of DNA and the cut vector are joined
together?
3. What is the final step in Recombinant DNA Technology?

Activity 2. Modeling Bacteria Transformation

Directions: Using the word choices provided in the boxes, fill in the numbered
boxes with the steps of bacteria transformation and the lettered lines with the
name of the structure next to them.

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Source:https://www.teachengineering.org/activities/view/uoh_genetic_lesson01_a
ctivity1.
Word Choices for Letters Word Choices for Numbers
foreign DNA with the bacteria transformed with recombinant
desired plasmid
gene plasmid cut with restriction enzyme
plasmid DNA ligase joins sticky ends to form
recombinant DNA recombinant plasmid

Guide Questions
1. What is the role of restriction enzymes in Recombinant DNA Technology?
2. What is the function of the DNA Ligase?
3. What is a Plasmid?

Activity 3 . Recombinant DNA Technology

Directions: Read the choices from each numbered item in Column A and identify
which is NOT included from these groups. Then classify it by choosing the
correct answer in Column B.
Note: To get one (1) point from this activity, two (2) responses must be
answered correctly.

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 Column A Column B
1. a. Primers A. Isolation of Genetic Material
b. DNA Polymerase
c. Agarose Gel B. Restriction Enzymes
d. PCR Digestion

2. a. Transformation
b. Ligation C. Amplification Using PCR
c. Recombinant DNA
d. Ligase D. Ligation of DNA Molecules

3. a. Gel Electrophoresis
b. Protein expression E. Insertion of Recombinant
c. Positive electrode DNA into Host
d. Agarose Gel
F. Isolation of Recombinant
4. a. Marker gene is employed. Cells
b. Filtering of the transformed host
cell. G. Obtaining or culturing the
c. Negatively charged DNA travels to Foreign Gene product
the positive electrode.
d. Mixed population of transformed
and non-transformed cells.

5. a. DNA is separated based on size.

b. Cutting out of digested DNA
fragments.
c. Negatively charged DNA travels to
the positive electrode.
d. Amplify a single copy of DNA into
thousands or millions

Guide Questions
1. What are Primers?
2. What is the function of the PCR in Recombinant DNA Technology?
3. What is the charge of the DNA?

Activity 4. Complete the Steps in Recombinant DNA Technology

Directions: Complete the figure below by supplying the missing Step in
Recombinant DNA Technology.
1. Isolation of Genetic Material.

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 2.

3. Amplification Using PCR.

4.

5.

6. Isolation of Recombinant Cells

7.

Guide Questions
1. What enzyme is used in DNA Ligation?
2. In what step in Recombinant DNA does Transformation occur?
3. What is the function of the Gel Electrophoresis in Recombinant DNA
Technology?

Activity 5. Describing the steps in Recombinant DNA Technology

Directions: Identify which step in Recombinant DNA Technology is involved.
Event Steps in Recombinant DNA
Technology
1. Running out the DNA on an agarose
gel.
2. Harboring cloned genes of interest
are grown on small a scale in the
laboratory.
3. DNA must be separated and
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 purified.
4. The recombinant DNA is introduced
into a recipient host cell
5. Making multiple copies of a DNA
sequence
Guide Questions
1. What equipment makes multiple copies of the DNA?
2. How do DNA fragments separate in Gel Electrophoresis?
3. What enzyme is being used to separate and purify the DNA from the cell?

Activity 6. Application of Recombinant DNA Technology

Directions: Read and understand the situation below about the Humulin R.
Answer the questions below after reading the article.

Eli Lilly began producing insulin from animal pancreas but fell short of the
demand, and the potency varied up to 25% per lot. The development of an
isoelectric precipitation method led to purer and more potent animal insulin,
decreasing the variation between lots to 10%. These discoveries led to the
introduction of longeracting animal insulins in the market. Protamine zinc insulin
lasted 24–36 hours. Isophane neutral protamine Hagedorn lasted 24 hours and
could be mixed with regular insulin. The pharmacokinetics and effects of
amorphous lente insulin (semilente, lente, and ultralente) depended on the
proportion of zinc. In 1978, the first recombinant DNA human insulin was
prepared by David Goeddel and his colleagues (of Genentech) by utilizing and
combining the insulin A- and B- chains expressed in Escherichia coli. Thereafter,
Genentech and Lilly signed an agreement to commercialize rDNA insulin. In
1982, the first insulin utilizing rDNA technology, Humulin® R (rapid) and N (NPH,
intermediate-acting), were marketed. Guide Questions
1. Are you in favor of using the animal pancreas to replace the insulin in the
human body? Why?
2. Based on the article, how does Recombinant DNA benefit humans?
3. What is Recombinant DNA Technology?
4. What is the function of a vector?
5. What are the common vectors in Recombinant DNA
Technology?

What I Have Learned

Directions: Fill in the blanks to complete the statements.

1. The general name for a piece of DNA that has been created by the
combination of at least two strands of DNA is called _____________.
2. The process of introducing recombinant DNA into a recipient host cell is
called _____________ .
3. Agarose Gel Electrophoresis involves running out the DNA on an
_____________.
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 4. The process of joining the cut fragment of DNA and the cut vector together
using an enzyme is called_____________ .
5. The _____________ is a method of making multiple copies of a DNA
sequence using an enzyme.
6. Recombinant DNA technology refers to the joining together of
_____________ from two different _____________ that are inserted into a host
organism to produce new genetic combinations.
7. Ligation of DNA molecules uses_____________ to cut the vector.
8. An enzyme called _____________ helps to amplify a single copy or a few
copies of DNA into thousands to millions of copies.
9. PCR reactions are run on _____________ to amplify a single copy or a few
copies of DNA.
10.The small circular molecules which act as carriers for the DNA fragments
are called _____________.

What I Can Do

Activity 1. Recombinant DNA Technology in our Life

Directions: By giving examples, explain the importance of Recombinant DNA
Technology in the given fields.

1. Health
2. Food
3. Environment

Assessment

Directions: Read each statement carefully. Choose the letter of the correct
answer.
1. Which enzyme is being used to amplify the DNA?
a. Endonuclease
b. DNA Helicase
c. DNA Ligase
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 d. DNA Polymerase

2. The following statements are all TRUE EXCEPT

a. The DNA is located in the nucleus of the cell.
b. Recombinant DNA is a combination of two different strands of DNA.
c. DNA Ligase is used to amplify a single copy or a few copies of DNA.
d. In Agarose Gel Electrophoresis the negatively charged DNA travels
to the positive electrode and is separated out based on size.

For question nos. 3-9, Arrange in order the steps in Recombinant DNA
Technology.
Use letters a to g
3. Amplification Using PCR
4. Isolation of Genetic Material
5. Insertion of Recombinant DNA into Host 6. Obtaining or culturing the
Foreign Gene product.
7. Ligation of DNA Molecules.
8. Restriction Enzymes Digestion
9. Isolation of Recombinant Cells

10. Which of the following refers to chemically synthesized oligonucleotides

that are complementary to a region of the DNA?
a. Agarose Gel
b. Primers
c. Restriction Enzymes
d. Vectors

11. Which of the following is TRUE?

a. DNA is positively charged.
b. Ligase is used to isolate the genetic material.
c. Thermal Cycler cuts fragment of DNA and the vector.
d. Gel Electrophoresis separates the DNA Molecule according to size.

For question nos. 12-15. Identify the steps in Recombinant DNA Technology that
are being described in each statement. The choices are as follows:

a. Amplification Using PCR

b. Isolation of Genetic Material
c. Insertion of Recombinant DNA into Host
d. Obtaining or culturing the Foreign Gene product.
e. Ligation of DNA Molecules.
f. Restriction Enzymes Digestion
g. Isolation of Recombinant Cells

12. Marker gene of plasmid vectors is employed.

13. The aim is to produce a desirable protein expression.

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 14. The DNA molecule is separated and purified using enzymes. 15. The
recombinant DNA is introduced into a recipient host cell.

Additional Activity

Activity 1.Implications of Recombinant DNA Technology

Explain how recombinant DNA Technology poses a threat to a population or
ecosystem.

Rubric for the Essay

Category 4 3 2 1

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 Reflective The idea The idea The idea The idea does
Thinking explains explains attempts to not address the
the the demonstrate student’s
student’s own student’s thinking about thinking and/or
thinking and thinking about learning but is learning.
learning his/her own
vague and/or
processes, as learning
unclear about
well as processes.
implications the personal
for future learning
learning. process.
Analysis The idea is an The idea is The idea The idea does
in-depth an analysis of attempts to not move
analysis of the learning analyze the beyond a
the learning experience learning description of
experience, and the value experience the learning
the value of but the value
of the derived experience.
the derived of the
learning to
learning to learning to
self or others, self or others.
the student or
and the
others is
enhancement
of the vague and/or
student’s unclear.
appreciation
for the
discipline.
Making The idea The idea The idea The idea does
Connections articulates articulates attempts to not articulate
multiple connections articulate any connection
connections between connections to other
between this between learning or
this learning this
experiences.
learning experience learning
experience and content experience
and content from past and content
from past learning from past
learning, experiences, learning
life and/or future experiences,
experiences goals. or personal
and/or future goals, but the
goals. connection is
vague and/or
unclear.

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 some organisms.
could lead Technology
to extinctionmight
of change the gene frequency of a population that
Introduction of genetically modified organisms which are product of Recombinant DNA
Possible answer:
Answers may vary
Additional Activity

Vector 10.
.B 10 Cycler
Thermal 9.
.F 9 polymeraseDNA 8.
harm to the environment
DNA ligase 7.
.B pesticide,
8 therefore lessuses less
species
.D Pest
7 resistant cropsEnvironment
-
molecules,DNA 6.
.G 6 enhanced nutritional value. PCR 5.
. C 15 .E 5 Golden Rice with a Food - Ligation 4.
. B 14 .A 4 helps Diabetic people. gel
Agarose 3.
. D 13 .C 3 Artificial human insulin Health
- Transformation 2.
. G 12 .C 2 Possible answer: DNA
. A 11 .D 1 Answers may vary Recombinant 1.
Assessment What Can I Do What I Have Learned

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