Module 2 – DNA Recombination and DNA Technology

DNA Recombination and DNA Technology

Module 2

EXPECTED LEARNING OUTCOMES

2.1 Define recombinant DNA technology and explain how it is used to clone genes and
manipulate DNA.
2.2 Describe how agarose gel electrophoresis, polymerase chain reaction, DNA
sequencing, and other molecular techniques are used to study gene structure,
function, and expression.
2.3 Understand major findings of the Human Genome Project, potential scientific and
medical applications of knowledge about the human genome, and associated
ethical, legal, and social issues.

THE BIG IDEA

The breakthrough in life sciences resulted in the creation of a genetic approach that is still
widely used. Agriculture, the food industry, pharmaceuticals, and several medical applications are only
a few examples. While biotechnology has been used for thousands of years in applications such as
winemaking and breadmaking, as well as selective breeding, it was only after Watson and Crick
discovered the DNA structure that this technique came into existence.

When scientists James Watson and Francis Crick discovered that DNA is a double-helical
molecule, they speculated at the discovery's possible relevance and effect. And these Nobel Laureates
could not have expected the unprecedented pace at which molecular biology would progress over the
next half-century.

The use of organisms and their materials in the development of essential products is known
as biotechnology. In vitro genetic experimentation revolutionized genetics by allowing scientists and
researchers all over the world to manipulate and engineer genes and gene products of every organism,
which has now become the cutting edge of modern biotechnology.

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DEEPEN YOUR UNDERSTANDING

Recombinant DNA technology is a form of genetic manipulation in which a foreign gene is

introduced into a gene of a particular organism in order to modify or strengthen it. In genetic
engineering, recombinant DNA or recombinant plasmid refers to plasmid DNA (circular DNA found in
prokaryotes) that has been incorporated into a gene of interest derived from another organism.

Recombinant DNA has

proved to be extremely crucial as
it determines gene function in a
number of biological processes,
including homeostasis,
reproduction, and growth. This
technology increases crop yields,
the consistency of fruits and
vegetables in terms of quantity,
weight, and nutritional content,
and the by-product of farmed
animals in food and agriculture.

Recombinant DNA is also Recombinant DNA technology

used in medicine, primarily for the
treatment and diagnosis of chromosomal disorders. Another use of this technology is Molecular
Medicine, which uses inferred amino acid sequences to improve drug design. Protein-based
medications and supplements are being developed at an alarming pace. It will, for example, inject an
insulin gene from a mammalian source into bacteria, causing them to multiply a thousand fold. These
bacteria will now synthesize insulin because they have the insulin gene, making them a biochemical
factory.

Genetic Engineering and Recombinant DNA Technology

Constructing a recombinant plasmid is the first step in engineering an organism. This is

accomplished by identifying and isolating a gene of interest from a source organism. It uses restriction
endonucleases to cut the target gene from the source organism's linear DNA. Different nucleotide
sequences are targeted by these enzymes. Meanwhile, it extracts plasmid DNA (a circular DNA
molecule isolated from the large chromosome in bacteria) from a bacterium, normally E. coli.

After which, it cuts the plasmid using restriction endonuclease at a certain location. Both the
gene of interest and the plasmid previously cut have sticky ends on the cut portion allowing them to
attach to each other. It needs the enzyme DNA ligase to help the two DNA join, making a circular
construct. At this point, the construct is now the recombinant plasmid. It allows E. coli to take up the
recombinant plasmid produced in a culture medium via the process of transformation. The bacteria
multiply along with the recombinant plasmid it contains. This process of making a construct and
allowing multiplication of E. coli containing recombinant plasmids is called gene cloning because the
products that have been producted contains the very samegenetic material introduced into the
bacterial plasmid. To ensure if the E. coli has taken up the recombinant plasmid, it also inserts both

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reporter genes and antibiotic resistance gene in the construct along with the gene of interest. If it
supplements antibiotic to the growing medium, only the E. coli cells containing the recombinant
plasmids will be able to grow.

Transgenic Organisms

While bacteria can successfully generate some recombinant proteins used in medicine, certain
proteins need a eukaryotic animal host for proper processing. The desired genes are then cloned and
expressed in animals such as sheep, goats, ducks, and mice. Transgenic animals are those that have
been genetically engineered to produce recombinant DNA. Several human proteins are expressed in
the milk of transgenic sheep and goats, as well as in the eggs of transgenic chickens. Mice have been
widely used in the expression and study of recombinant genes and mutations.

Manipulation of plant DNA (i.e., genetically modified organisms or GMOs) has aided in the
development of favorable traits like disease tolerance, herbicide and pesticide resistance, improved
nutritional performance, and longer shelf life. Humans depend on plants for their primary source of
nutrition. Years before industrial biotechnology practices, farmers devised methods for selecting plant
varieties with desirable traits.

Transgenic plants are those that have obtained recombinant DNA from another species. Since
they are not natural, government authorities closely track transgenic plants and other GMOs to ensure
that they are safe for human use and do not damage other plant and animal life. To ensure ecological
stability, rigorous testing is needed since foreign genes will spread to other organisms in the world.
The first crop plants to be genetically modified were staples like rice, potatoes, and tomatoes.

• Transformation of Plants Using Agrobacterium tumefaciens

In microbial organisms, gene

transfer happens spontaneously
between animals. The DNA of several
viruses that cause human diseases is
integrated into the human genome.
The bacterium Agrobacterium
tumefaciens causes tumors in plants
by moving DNA from the bacterium
to the cell. The tumors do not kill the
plants, but they do stunt them and
make them more vulnerable to harsh
Recombinant DNA technology
environmental conditions. A.
tumefaciens affects a variety of
species, including walnuts, apples, nut trees, and beets. Because of the thick plant cell wall,
artificial DNA introduction into plant cells is more difficult than in animal cells.

To insert DNA fragments of their choosing into plant hosts, researchers used the natural
transition of DNA from Agrobacterium to a plant host. The pathogenic A. tumefaciens has a set of
plasmids called Ti plasmids (tumor inducing plasmids) that contain genes for the formation of
tumors in plants in nature. The Ti plasmid's DNA is inserted into the genome of the contaminated
plant cell. The tumor-causing genes are removed from Ti plasmids, and the desired DNA fragment
is inserted for transition into the plant genome. Antibiotic resistance genes are carried on the Ti
plasmids to aid selection, and they can also be propagated in E. coli cells.

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• The Organic Insecticide Bacillus thuringiensis

Bacillus thuringiensis (Bt) is a bacterium that
creates protein crystals during sporulation that are
poisonous to many insect species that invade plants. In
order for Bt toxin to be triggered, insects must consume
it. Within a few hours, insects that have consumed Bt
toxin have stopped feeding on the plants. Death begins
just a few days after the toxin is released in the insects'
intestines. Plants will now encrypt their own crystal Bt
toxin that destroys insects due to advanced
biotechnology. Plants have been given the crystal toxin
genes that were cloned from Bt.

• Flavr Savr Tomato

In 1994, the first GM crop was planted. It was a
tomato that was immune to decay and had a longer shelf
life. Antisense RNA technology was used to delay the
softening and rotting of GM tomatoes caused by fungal
infections, resulting in a longer shelf period for the GM
tomatoes. This tomato's taste was improved by further
genetic engineering. Due to issues with seed
maintenance and distribution, this GM tomato was
unable to remain on the market.

Bt corn

Basic Techniques to Manipulate Genetic Material

Remember that nucleic acids are macromolecules made up of nucleotides (a sugar, a

phosphate, and a nitrogenous base) bound through phosphodiester bonds to consider the
conventional approaches used to deal with them. Each of these molecules' phosphate groups has a
net negative charge. The genome refers to the whole set of DNA molecules in the nucleus. DNA is
made up of two strands that are joined by hydrogen bonding between the paired bases. Exposure to
high temperatures (DNA denaturation) will split the two strands, and can then be reannealed by
cooling. The DNA polymerase enzyme can duplicate the DNA. Unlike DNA, which is located in the
nucleus of eukaryotic cells, RNA
molecules leave the nucleus. The
messenger RNA (mRNA) is the most
commonly studied type of RNA since
it represents actively expressed
protein coding genes. However,
since RNA molecules are less stable
than DNA, they pose significant
challenges to study.

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DNA and RNA Extraction

The DNA or RNA must first be separated or removed from the cells in order to analyze or
modify nucleic acids. Conventional techniques are used to remove various forms of DNA (Figure 17.2).
Most nucleic acid extraction procedures require steps to break open the cell and use enzymatic
reactions to remove any unnecessary macromolecules (for example, degradation of unwanted
molecules and separation from DNA sample). A lysis buffer (a solvent that is acidic) is used to break
down cells since the term lysis means "to break." Lipid molecules in cell membranes and nuclear
membranes are torn apart by these enzymes. Proteases, which break down proteins, and
ribonucleases, which break down RNA, are enzymes that inactivate macromolecules. Alcohol is first
used to precipitate the DNA. Human genomic DNA appears as a gelatinous, white mass in most cases.
The DNA samples can be held frozen for many years at –80°C.

Gene expression variations in cells are studied using RNA analysis. Since RNAses are abundant
in nature and difficult to inactivate, RNA is naturally very unstable. RNA extraction, like DNA
extraction, requires a combination of buffers and enzymes to inactivate macromolecules while
retaining the RNA.

Gel Electrophoresis

Nucleic acids may be

mobilized by an electric field
since they are negatively
charged ions at neutral or basic
pH in an aqueous setting. Gel
electrophoresis is a method that
uses electric charge to separate
molecules based on their size.
Whole chromosomes or
fragments of chromosomes may
be isolated from nucleic acids.
Agarose Gel Electrophoresis
The nucleic acids are inserted
into a slot near the negative electrode of a semisolid, porous gel matrix and dragged toward the
positive electrode at the other end. Smaller molecules migrate quicker through the pores in the gel
than larger molecules; this variation in migration rates distinguishes the fragments based on size.

To have a size reference, molecular weight standard samples can be run alongside the
molecules. Various fluorescent or colored dyes may be used to observe nucleic acids in the gel matrix.
On the basis of their size, distinct nucleic acid fragments occur as bands at unique distances from the
top of the gel (the negative electrode end). Uncut genomic DNA is normally too large to run through
the gel and forms a single large band at the top of the gel, while a mixture of genomic DNA fragments
of different sizes appears as a long smear.

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Polymerase Chain Reaction

When genomic DNA is

collected in bulk, it is visible to the
naked eye, but DNA analysis often
requires concentrating on one or
more unique regions of the
genome. The polymerase chain
reaction (PCR) is a technique for
amplifying complex DNA regions for
further investigation. In
laboratories, PCR is used for a
variety of tasks, including cloning
gene fragments to study genetic
Polymerase Chain Reaction
disorders, detecting contaminant
foreign DNA in samples, and amplification of DNA for sequencing. The diagnosis of hereditary diseases
is one of the most practical applications of PCR.

Reverse transcriptase PCR (RT-PCR) can also be used to amplify DNA fragments from an RNA
template. The first step is to use DNA nucleotides to replicate the initial DNA template strand (called
cDNA) from mRNA. This is referred to as reverse transcription. This generally requires the existence of
a reverse transcriptase enzyme. After the cDNA has been produced, it can be amplified using standard
PCR.

Genetic Engineering

The use of recombinant DNA technologies to alter an organism's DNA to produce beneficial
characteristics is referred to as genetic engineering. The most popular method of genetic engineering
is the insertion of foreign DNA in the form of recombinant DNA vectors produced by molecular cloning.
A genetically modified organism (GMO) is one that has been given recombinant DNA. The host
organism is referred to as transgenic if the foreign DNA added is from a different species. Since the
early 1970s, bacteria, plants, and livestock have been genetically engineered for research, medicinal,
agricultural, and industrial uses. GMOs, such as herbicide-resistant soybeans and borer-resistant corn,
are used in many packaged foods in the United States.

Genetic Diagnosis and Gene Therapy

Genetic diagnosis by genetic testing is the practice of testing for possible genetic
abnormalities before administering medication. Family members may be recommended to seek
genetic tests based on the inheritance patterns of a
disease-causing gene. Women with breast cancer, for
example, are often recommended to undergo a biopsy
so that the surgical team can assess the hereditary
cause of the cancer's origin. The results of genetic
studies that decide the type of cancer are used to
develop treatment strategies. If the cancer is affected
by hereditary gene mutations, other female relatives
should be tested and screened for breast cancer on a
regular basis. In families of particular chronic

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conditions, genetic testing for fetuses (or embryos with in vitro fertilization) is now available to assess
the presence or absence of disease-causing genes.

Gene therapy is a disease-curing method based on genetic modification. In the most simplistic
nature, it means adding a healthy gene at a random position in the genome to help treat a disease
caused by a defective gene. The good gene is normally delivered to diseased cells via a virus vector
that infects the host cell and delivers the foreign DNA. More recent models of gene therapy, such as
those used to treat severe combined immunodeficiency (SCID), attempt to fix the mutation at the
initial site in the genome.

Production of Vaccines, Antibiotics, and Hormones

To mass generate the target antigen, existing

techniques use microorganism genes cloned into Gene therapy (adenovirus vector)
vectors. The antigen is then injected into the bloodstream, stimulating the primary immune response
and triggering immune memory. Genes cloned from influenza virus strains have been used to treat
the virus's ever-changing strains.

Antibiotics are a product of biotechnology. Microorganisms, such as fungi, develop them

spontaneously to achieve an edge over bacterial populations. Antibiotics are mass-produced by
growing and manipulating fungal cells on a massive scale.

As early as 1978, recombinant DNA technology was used to generate vast amounts of human
insulin in E. coli. Previously, diabetes could only be controlled with pig insulin, which, due to variations
in the gene product, triggered allergic reactions in humans.

LEARNING RESOURCES

Books

Thieman, W. J., & Palladino, M. A. (2019). Introduction to biotechnology, global edition.

Pearson Education Limited.

Franceschetti, D., & Crawford, C. A. (2018). Principles of biotechnology. Amsterdam

University Press.

Thieman, W. J., & Palladino, M. A. (2016). Introduction to biotechnology (3rd edition) (3rd

ed.). Pearson.

Clark, D. P., & Pazdernik, N. J. (2015). Biotechnology. Elsevier Gezondheidszorg.

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Ratledge, C., & Kristiansen, B. (2013). Basic biotechnology (Third ed.). Cambridge

University Press.

Images

Anderson, W. (2019). BT Corn [Image]. School Work Helper. https://schoolworkhelper.net/bt-

corn-genetically-modified-corn/

AMGEN Foundation. (2021). Gel electrophoresis [Illustration]. Khan Academy.

https://www.khanacademy.org/science/ap-biology/gene-expression-and-
regulation/biotechnology/a/gel-electrophoresis
Aryal, S. (2018, September 19). Recombinant DNA technology [Illustration]. MIcrobe Notes.
https://microbenotes.com/recombinant-dna-technology-steps-applications-and-limitations/

Biotechnology and how it changed our lives. (2018, December 11). [Image]. Nuclineers.
https://nuclineers.com/whats-biotechnology/

Editors of Encyclopedia Britannica. (2019, April 18). PCR [Diagram]. Encyclopedia

Britannica. https://www.britannica.com/science/polymerase-chain-reaction

Genetic engineering in plants. (2020, December 17). [Image]. 78 Steps Health.

https://www.pinterest.ph/pin/438678819955742690/

Zedalis, J., Eggebrecht, J., Rye, C., & Wise, R. (2018). DNA extraction [Diagram]. In
OpenStax AP Biology (p. 689).
Singh, B. (2014). Gene theraphy [Image]. SciNews. http://www.sci-
news.com/medicine/science-retooling-human-gene-therapy-new-adenoviral-vectors-02109.html

ABOUT MODULE

Module Creator/Curator: Ms. Larisa Mae C. Agtay

Template and Layout Designer: Mr. Florence Somoria

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