Republic of the Philippines

DEPARTMENT OF EDUCATION
Region IV-A CALABARZON
PROVINCE OF BATANGAS
St. Blaise Community Academy, Inc.
San Luis, Batangas/ [email protected]
 043 – 740-960609997646638 / 09178076741

Name of Student: _______________________________________________ Section: _______________________

Present Address: ________________________________________________ Contact no.______________________
Subject Matter: GENERAL BIOLOGY 2
Topic: RECOMBINANT DNA (Week 2)
WHAT YOU ARE SUPPOSED TO LEARN?
After going through this lesson, you should be able to:
1. give examples of products from recombinant DNA technology.
2. illustrate the use of databases to search genes for desired traits.
3. describe steps in PCR to amplify and detect a gene of interest.
WHAT I KNOW?

Direction: Read and analyze the following questions, encircle the letter of the correct answer.
1. Cutting DNA from one organism and placing it into the DNA strand of another organism is called?
a. recombinant RNA technology b. selective breeding
c. recombinant DNA technology d. gene cloning
2. A __________ is required to transfer genes from one organism to another.
a. vector b. transport molecule c. reverse transcriptase d. genetic probe
3. In order to insert a human gene into plasmid, both must.
a. code for the same gene product b. be cut by the same restriction enzyme
c. originate from the same type of cell d. have identical sequences
4. What enzyme forms covalent bonds between restriction fragments?
a. DNA primase b. DNA ligase c. DNA helicase d. DNA polymerase
5. An organism in which foreign genes have been incorporated is called a ________?
a. recombinant organism b. polymorphism
c. transgene recombinant d. transgenic organism

WHAT’S NEW?

Activity: Applications of your knowledge.

Directions: Try to guess what applications of recombinant DNA illustrate the following pictures. Write your answer on your activity
notebook.

PRESENTATION OF RECOMBINANT DNA

ENVIRONMENT- With increasing world’s population

at a greater rate, human requirements for food are rapidly increasing.
Rapid increase industrialization has soared up the environmental pollution
and industrial wastes are directly allowed to mix with water, which has affected aquatic marines and indirectly, human beings.
Therefore, these issues urge to be addresses through modern technologies.
DNA technology has led to other advances in the creation of genetically modified crops and identification and treatment of genetic
diseases.
Biotechnology is the manipulation of organisms or their components to make useful products and has been used for thousands of
years to make bread using yeast and selectively breed livestock for desired traits. Biotechnology today means the use of DNA
technology techniques for studying and manipulating genetic material and modifying specific genes.
RECOMBINANT DNA TECHNIQUES
Bacteria are the workhorses of modern biotechnology. To work with genes in the laboratory, biologists often use bacterial
plasmids, small, circular DNA molecules that replicate separately from the larger bacterial chromosome.
DNA LIGASE is an enzyme that seals the bonds between restriction fragments.
The purified DNA and the vector of interest are cut with the same restriction enzyme.
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.
In gene cloning, the original plasmid is called a cloning vector. A cloning vector is a DNA molecule that can carry foreign
DNA into a host cell and replicate there. Bacterial cells do not accept foreign DNA easily. Therefore, they are treated to make them
compotent to accept new DNA. The processes used may be thermal shock, Ca++ ion treatment
Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction or PCR is a method of making multiple copies of 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. A three-step cycle:
heating, cooling and replication- brings about a chain reaction that produces an exponentially growing population of identical DNA
molecules. The key to PCR is an unusual heat stable DNA polymerase called Taq polymerase.

Applications of Recombinant DNA Technology

Many fields benefit from DNA technology and genetic engineering these are found in:
 Medical Applications
 Pharmaceutical Products
 Forensic Evidence and Genetic Profiles
 Environmental Clean-up
 Agricultural Applications

Limitations of Recombinant DNA technology

 Destruction of native species in the environment the genetically modified species are introduced in
 Resilient plants can theoretically give rise to resilient weeds which can be difficult to control
 Cross contamination and migration of proprietary DNA between organisms
 Recombinant organisms contaminating the natural environment
 The recombinant organisms are population of clones, vulnerable in exact same ways. A single disease or pest can wipe out
the entire population quickly
 Creation of superbug is hypothesized
 Ethical concern about humans trying to play God and mess with the nature’s way of selection. It is exaggerated by the fear of
unknown of what all can be created using the technology and how is it going to impact the civilization
 Such a system might lead to people having their genetic information stolen and used without permission
 Many people worry about the safety of modifying food and medicines using recombinant DNA technology

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 RECIPIENT ORGANISM APPLICATION (FIELD)

Insulin Production Insertion of Human Insulin Bacteria (Medicine)Production of
Gene Human Insulin in Bacteria
Pest Resistance Insertion of Bt-toxin gene Corn/Maize (Agriculture) Production of
corn
Delayed Ripening Disruption of a gene for a Tomato plant (Agriculture) Production of
ripening enzyme (e.g., plants with fruits that have
polygalacturonase) delayed ripening fruits. These
fruits will survive longer
transport time, allowing their
delivery to further locations
(i.e. Export deliveries
Chymosin Production Insertion of a gene for Bacteria (Industry) Enhance large scale
chymosin production of chymosin. This
enzyme serves as a substitute
for rennet in the coagulation
of milk. Rennet must be
harvested from calves. The
large-scale production of this
enzyme in bacteria provides
an abundant supply of this
important component.

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;
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)
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 the coding 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.
1. 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.

WHAT’S MORE?
Activity 1: Answer the following questions in not less than 5 sentences..

1. Explain the concept of Recombinant DNA Technology and its importance.

2. Differentiate the three basic steps in PCR Amplification.
3. Cite atleast 3 limitations of Recombinant DNA Technology and elaborate it.

WHAT I CAN DO?

Activity 2: Make a poster/slogan showing the application of recombinant DNA either in the field of Agriculture, Medicine and Food
Industry.

ASSESSMENT

Direction: Identify what is being described in the following statements.

________________1. It involves manipulating genes in order to make useful products.
________________2. It is used to carry a desired DNA sequence into a host cell.
________________3. It is a small circular DNA found in bacteria.
________________4. It is the process of heating the reaction strongly to separate the DNA strands.
________________5. It is used to cut the plasmid at specific sites.
________________6. Raise the reaction temperatures so taq polymerase can extend its primers.
________________7. Cool the reaction so the primers can bind to their complementary sequences.
________________8. It is the term for heat resistant DNA polymerase.
________________9. A method of making multiple copies of DNA sequence using the enzyme- DNA polymerase in vitro.
________________10. Short term for Thermos Acquatic.

WHAT CAN I SHOW?

Explain in 5 sentences the quotation below.

DNA is like a computer program but far more advanced than any software ever created.
ANSWER KEY

WHAT I KNOW?

1. C

2. A

3. B

4. B

5. D

WHAT’S NEW?

1. Bt corn/ pest resistance

2. For human growth hormone

3. Modify food e.g. sweetness and color

4. Nutritional Improvement

ASSESSMENT

1. Genetic Engineering

2. Vector

3. Plasmid

4. Denaturation

5. Restriction Enzyme

6. Extension

7. Annelaing

8. Taq Polymerase

9. PCR

10. Taq
 References:
https://blogs.scientificamerican.com/observations/the-concept-
of-race-is-a-https://flexbooks.ck12.org/cbook/ck-12-middle-
**THIS PORTION OF THE MODULE SHOULD BE SUBMITTED BACK TO SBCA FOR CHECKING**
school-life-science
Name of Student: ______________________________________________ 2.0/section/3.18/primary/lesson/recombinant-dna-https://www.
Present Address: _______________________________________________ ck12.org/book/cbse_biology_book_class_xii/section/14.1/
Contact no.____________________
Subject Matter: GENERAL BIOLOGY 2 https://www.ck12.org/section/dna-technology/
Topic: RECOMBINANT DNA (Week 2)
WHAT I KNOW: PRE-TEST
1. ___________ 2.________________ 3.________________ 4.____________ 5._______________

WHAT’S MORE: ANALYSIS

WHAT I CAN DO:

ASSESSMENT: POST-TEST

WHAT I CAN SHOW: ESSAY