LEARNING GUIDE 01 BASIC TECHNIQUES

Subject Code BIOLOGY 2 Fundamentals of Biology II

Learning Guide 1.0 Basic Techniques
Lesson Code 1.2 Gel Electrophoresis and Polymerase Chain
Reaction (PCR)
Time Frame 30 minutes

MATERIALS NEEDED
To complete this learning guide, you need the following:
1. pen;
2. paper;
3. phone/tablet/laptop;
4. Adobe scanner mobile app;
5. Moodle Learning Management System account;
6. stable internet connection and;
7. Biology: A Global Approach by Campbell et al. (2018).
Time
TARGET Allotment
(1 min)
After completing this learning guide, you are expected to:

• describe the processes and explain their function in identifying DNA, RNA, and
HOOK proteins.

Time
Allotment
HOOK (5 min)

Let us review the processes in the Central Dogma

of Molecular Biology: DNA (gene) à RNA (transcript) à
Proteins (trait). Depending on their chromosomes, different
species have different characteristics (DNA sequences).
For instance, frogs have antimicrobial peptides on their
skin. There are proteins in some jellyfish that cause them
to shine in the dark (like the green fluorescent protein or
GFP). Mutations contribute to anemia in the hemoglobin
genes.

If transcription and translation of genes will result

This Photo by Unknown Author is licensed in particular characteristics, then the insertion of certain
under CC BY-SA. genes in a given organism could also give it new
characteristics. This is the basis of the development of
genetically modified organisms (GMOs).

In order to modify their properties, there are several different characteristics that can be applied
to organisms. Examples of changed traits using cloned genes and their applications are shown in the
following table:

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 Modified Trait Gene Modification Recipient Organism Application (Field)
Insulin Production Insertion of human Escherichia coli Bacterial production
insulin-producing gene (Bacteria) of human insulin
(Medicine)
Pest Resistant Crops Insertion of a Corn (Maize), Cotton Production of
particular Bt (Bacillus insecticidal proteins
thuringiensis) toxin that selectively kill a
gene few specific insect
species
(Agriculture)
Delayed Ripening Regulation of fruit Tomato Production of fruit
polygalacturonase (an plants that have
enzyme that increases delayed fruit ripening.
the ripening process) These fruits can
withstand longer
transport times,
enabling them to be
shipped to distant
locations
(Agriculture)

When a desired trait is selected, for either its identification or expression in a given organism,
a macromolecule is analyzed and purified using electrophoresis and information is acquired through
polymerase chain reaction (PCR). Gel Electrophoresis is performed using a special medium, most
often a gel that has been extensively developed for molecular separations of very large molecules such
as proteins and nucleic acids. On the other hand, Polymerase Chain Reaction (PCR) is an
amplification technique to produce thousands to millions of copies of DNA of interest by cloning the
unique or targeted sections of a DNA sequence.

Time
IGNITE Allotment
(25 min)
Agarose Gel Electrophoresis (AGE)
Electrophoresis is a technique for separating charged particles based on variations in their
migration rates. It is the method of applying an electric field through a mixture to move charged
molecules in solution. The charge, shape, and size of molecules affect their migration in an electric
field. Some media include paper, polyacrylamide or agarose gels which are the most common matrix
for electrophoresis of biological molecules.

Agarose Gel Electrophoresis (AGE) is a qualitative and semi-quantitative technique for

analyzing isolated DNA. DNA is “run” in AGE to separate fragments from each other, determine the
sizes of fragments and the presence or amount of DNA, and analyze restriction digestion products. AGE
is a separation technique based on mass where the agarose acts as a sieve. It typically resolves 200 bp
to 20 kbp and includes DNA size standards.

During electrophoresis, DNA molecules become entangled with obstacles in the agarose gel
matrix. The arms of the “U” point in the direction of the electric field in U-shaped configurations. One
of the arms eventually slips away from the barrier, and DNA collapses into a globular form.

Resolution of different sizes of DNA fragments is possible using gels with different
concentrations of agarose. Small DNAs can be separated more easily with higher agarose concentrations
while low agarose concentrations allow resolution of larger DNAs.

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 Figure 1.2a. AGE of DNA. This Photo by Unknown Author is licensed under CC BY.

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Polymerase Chain Reaction (PCR)
Detection

Some researchers may be interested in determining whether in a specific organism there is a

specified gene of interest or trait available for use. If this knowledge is not given by previous research,
researchers can test the DNA of different organisms for the presence of these particular genes. A
technique that allows particular genes to be identified in target species is called polymerase chain
reaction or PCR.

Figure 1.2b. DNA Amplification by PCR. This Photo by Unknown Author is licensed under CC BY-SA.

An in vitro method that simulates DNA replication in vivo is PCR amplification. It uses a
thermostable (heat-resistant) DNA polymerase that shapes unwound DNA templates with single
stranded DNA strands. To facilitate the unwinding (denaturation) of the DNA template (~95 °C); the
annealing of a primer (a ~20 bp oligonucleotide sequence (recall RNA primers in DNA replication)
onto the ssDNA template strand (~54 – 60 °C); and the elongation of the generated ssDNA strand by
attaching complementary bases to the template strand (~72 °C), PCR uses repeated incubation cycles
at different temperatures.

The polymerase’s thermal stability enables it to withstand repeated denaturation, annealing and
extension cycles with little enzyme function loss. The sum of the goal sequence is doubled by any cycle
of PCR. A standard PCR experiment uses approximately 35 amplification cycles. This increases by 235
(i.e. ~34 billion) times the original sum of the target sequence.

The detection of genes by PCR requires the design of primers that only bind to sequences
unique to a target. Researchers would like to find out, for example, whether gene X (such as the human
insulin-producing gene) is available in the target organism (e.g. a mouse, Mus musculus). Primers can
be built in the databases by looking at the available sequences for gene X (e.g. all the genes for insulin
in different organisms; humans, pigs, cows, etc.). Compared to matching areas of sequence similarity
(conserved sequences) and areas of sequence dissimilarity (non-conserved sequences), the various gene
X sequences must be aligned.

Primers can be classified as forward or reverse primers. Forward primers are complementary
and bind to the gene’s reverse complementary sequence (non-coding). Reverse primers complement
and bind to the gene’s coding sequence.

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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 ß CCATAGATC (Reverse Primer)

5’ GCGATGAGG 3’ à 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 or 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.

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New Strand 1:
Nucleotide # 3
Nucleotide # 39
37 bp product


5’ A T GCGATGAGGATATGACCCGATAGATAGAGGTATCTAGAGAT 3’ (Coding strand)
(old)
3’- CGCTACTCCTATACTGGGCTATCTATCTCCATAGATC – 5’ (Reverse Primer) (new)

New Strand 2:
Nucleotide # 3
Nucleotide # 39
37 bp product


5’ GCGATGAGGATATGACCCGATAGATAGAGGTATCTAG -3’ (Forward Primer) (new)
3’ T A C GCTACTCCTATACTGGGCTATCTATCTCCATAGATC TCTA 5’ (Non-coding strand)
(old)

PCR Applications

To detect the presence of a desired gene in an organism, PCR can be used. The expected product
could reflect either a particular region of the gene or the entire gene itself, depending on the primer
design. The first case is useful for gene identification or the detection of species inside a sample with
that particular gene. The second case is useful for the replication of the whole gene in other species for
eventual expression. The direct amplification or copying of a complete gene is part of that gene’s
“cloning” process.

For the NAVIGATE and KNOT components, refer to Learning Guide 1.3.

REFERENCES
Campbell, N., Reece, J., Mitchell L., & Taylor, M. (2003). DNA Technology and the Human Genome.
In N. Campbell, J. Reece, L. Mitchell, & M. Taylor, Biology Concepts & Connections (pp. 230-
279). Philippines: Pearson Education (Asia) Pte, Ltd.
Ramirez, D. (1991). Genetic Engineering and Biotechnology. In D. Ramirez, Genetics (pp. 181-193).
UPLB: SEAMEO SEARCA.
Reece, J.B., Urry, L.A., Cain, M.L., Wasserman, S.A., Minorsky, P.V., and Jackson, R.B. (2012).
Campbell Biology, (9th ed). The Benjamin Cummings Publishing Co., Inc.
Teaching Guide for Senior High School. General Biology 2. (2016). Discuss the Applications of
Recombinant DNA (pp. 36-48). Published by the Commission on Higher Education.

Prepared by:

JOEL C. MAGDAY, JR.

Special Science Teacher III
PSHS-CAGAYAN VALLEY CAMPUS

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Reviewed by: Approved by:

VIRNA JANE M. NAVARRO MICHELLE B. DUCUSIN

Special Science Teacher IV Special Science Teacher V/Team Lead (Bio)
PSHS-WESTERN VISAYAS CAMPUS PSHS-ILOCOS REGION CAMPUS

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