AN ASSIGNMENT

ON
RECOMBINANT DNA TECHNOLOGY

COURSE NO: SS-5203

COURSE TITLE: SOIL BIOTECHNOLOGY

SUBMITTED BY,

NASMIN NAHER

STUDENT ID: MS-211309

MS 1st YEAR, 2nd TERM

SOIL, WATER AND ENVIRONMENT DISCIPLINE

KHULNA UNIVERSITY

DATE OF SUBMISSION: 5th MAY, 2023

Principle: Recombinant DNA technology involves using enzymes and various laboratory
techniques to manipulate and isolate DNA segments of interest. This method combines (or
splices) DNA from different species or creates genes with new functions. The resulting copies
are often referred to as recombinant DNA. Such work typically involves propagating the
recombinant DNA in a bacterial or yeast cell, whose cellular machinery copies the engineered
DNA along with its own.

Recombinant DNA in a living organism was first achieved in 1973 by Herbert Boyer, of
the University of California at San Francisco, and Stanley Cohen, at Stanford University, who
used E. coli restriction enzymes to insert foreign DNA into plasmids.

The complete process of recombinant DNA technology includes multiple steps, maintained in a
specific sequence to generate the desired product.

Step-1. Isolation of Genetic Material.

The first and initial step in recombinant DNA technology is to isolate the desired DNA in its
pure form i.e. free from other macromolecules. 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 lysozymes, cellulase, chitinase,
ribonuclease, proteases etc. Other macromolecules are removable with other enzymes or
treatments. Ultimately, the addition of ethanol causes the DNA to precipitate out as fine threads.
This is then spooled out to give purified DNA.

Step-2. Cutting the gene at the recognition sites.

The restriction enzymes play a major role in determining the location at which the desired gene
is inserted into the vector genome. These reactions are called ‘restriction enzyme digestions’.
They involve the incubation of the purified DNA with the selected restriction enzyme, at
conditions optimal for that specific enzyme. 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

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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.

Step-3. Amplifying the gene copies through Polymerase chain reaction (PCR).

It is a process to amplify a single copy of DNA into thousands to millions of copies once the
proper gene of interest has been cut using restriction enzymes.

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.

The cut fragments of DNA can be amplified using PCR and then ligated with the cut vector.

Step-4. Ligation of DNA Molecules.

In this step of Ligation, the joining of the two pieces – a cut fragment of DNA and the vector
together with the help of the enzyme DNA ligase.

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 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.

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Step-5. Insertion of Recombinant DNA into Host.

In this step, the recombinant DNA is introduced into a recipient host cell. This process is termed
as 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, electroporation, etc. Once the recombinant DNA is inserted into the host cell, it
gets multiplied and is expressed in the form of the manufactured protein under optimal
conditions.

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Application of Recombinant DNA Technology

 DNA technology is also used to detect the presence of HIV in a person.

 Recombinant DNA is widely used in biotechnology, medicine and research.
 The most common application of recombinant DNA is in basic research, in which the
technology is important to most current work in the biological and biomedical sciences.
 Gene Therapy – It is used as an attempt to correct the gene defects which give rise to
heredity diseases.
 Clinical diagnosis – ELISA is an example where the application of recombinant DNA
was used.
 Recombinant DNA technology is widely used in Agriculture to produce genetically-
modified organisms such as Flavr Savr tomatoes, golden rice rich in proteins, and Bt-
cotton to protect the plant against ball worms and a lot more.
 In the field of medicines, Recombinant DNA technology is used for the production of
Insulin.
 Recombinant proteins are widely used as reagents in laboratory experiments and to
generate antibody probes for examining protein synthesis within cells and organisms.

Merits of recombinant DNA technology

Recombinant DNA technology, also known as "genetic engineering," has many advantages for
people. Recombinant DNA technology, for instance, was used to create synthetic human insulin.
People with diabetes are unable to make the insulin they require to process sugar on their own.
Since most people who use animal insulin experience severe allergic reactions, it is not a suitable
replacement. Because plasmids can replicate independently of chromosomes, scientists used
recombinant DNA technology to isolate the human insulin gene and insert it into them. After
being introduced into bacterial cells, these plasmids produced insulin using the genetic material
from human cells as a template. Humans could use the final insulin product without any risk. As
a result, after being diagnosed with diabetes, a person's life expectancy increased to that of a
healthy adult.

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Recombinant DNA technology helped improve food production. Fruits and vegetables, which
were prone to attacks from pests, now have genetic modifications to be more resistant. Some
foods have modifications for longer shelf lives or higher nutritional content. These advancements
greatly increased crop yields, which means that more food is available to the public at the end of
each growing cycle.

Scientists have been working to improve vaccines and produce new ones using recombinant
DNA technology. These "DNA vaccines," which utilize recombinant DNA, are in the testing
stages. Most modern vaccines introduce a small "piece" of a disease into the body, so the body
can develop ways to fight that particular disease. DNA vaccines would directly introduce the
antigen itself and lead to more immediate and permanent immunity. Such vaccines could
potentially protect people against diseases such as diabetes and even cancer.

Demerits of recombinant DNA technology

Most of the downsides of recombinant DNA technology are ethical in nature. Some people feel
that recombinant DNA technology goes against the laws of nature, or against their religious
beliefs, due to how much control this technology gives humans over the most basic buildings
blocks of life. There are also other ethical issues. Some people are concerned that genetic
material would become an expensive commodity if businesses can pay scientists to buy, sell, and
patent genetic material. People's genetic data might be taken and utilized without their consent
under such a system. It may sound odd, but such cases have already happened. In 1951, a
scientist used unique cells stolen from a woman named Henrietta Lacks to create an important
cell line (the HeLa cell line) which is still used in medical research today. Her family did not
know about her involuntary donation until after her death, and never received compensation, but
others have profited from the use of HeLa cells.

Recombinant DNA technology is being used to modify foods and medications, which has many
people concerned about its safety. Despite numerous studies suggesting that genetically modified
foods are safe, it is understandable why there might be such concerns. Some individuals are
concerned that excessive human genetic manipulation could lead to social issues.

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