Recombinant
DNA
Technolgoy
Esther Sparks, Bri'an Barrow,
Michaela Palmer, Brianna Walters,
Arielle Johnson
Presentation
Outline
What is Recombinant DNA Technology
Steps Involved In Recombinant DNA
Technology
Examples
What is
Recombinant
DNA
Technology?
WHAT IS RECOMBINANT DNA
TECHNOLOGY?
Recombinant DNA technology involves using
enzymes and various laboratory techniques to
manipulate and isolate DNA segments of
interest. This method can be used to combine
(or splice) DNA from different species or to
create genes with new functions.
WHAT IS RECOMBINANT DNA
TECHNOLOGY?
Recombinant DNA is the name
given to DNA formed after a
piece of DNA from a donor
organism is joined to a piece of
DNA from another organism. If it
is inserted into a bacterium, it
will replicate or clone itself and
as the bacterium multiplies, the
recombinant DNA will multiply.
The piece of DNA to be cloned is
combined with either a plasmid or the
DNA of a bacteriophage. Plasmids are
small circular pieces of DNA found in
bacteria. They are separate from the
bulk of the DNA and can replicate
independently of the rest of the DNA.
Bacteriophages are viruses which can
‘inject’ their DNA into bacteria for
replication. The plasmid or
bacteriophage is known as the vector or
cloning vector, because it acts as a
carrier for the DNA to be cloned.
Steps Involved
in
Recombinant
DNA
Technology
1) DNA ISOLATION
Isolation of DNA is an enzymatically controlled process
where the plant or animal cells are treated with certain
enzymes.
Firstly the cell membrane of the cell with the desired
gene is broken using a lysis buffer. This process is known
as cell lysis. Afterwards, the DNA is extracted in its
purest form, meaning it is devoid of macromolecules
such as RNA, polysaccharides, proteins and lipids.
Because DNA coexists with these macromolecules it is
necessary for it to be purified using the enzymes such as
ribonucleases, proteases and lipases.
After these macromolecules are removed, the DNA
sample is then washed in isopropanol and then in
ethanol and it precipitates out as fine threads as a
result of the presence of ethanol.
In terms of insulin production, the gene for human
insulin is isolated in this way.
2) CUTTING OF THE DNA/ RESTRICTION
ENZYME DIGESTION
The isolated and purified DNA is treated with
restriction endonucleases which cut the DNA into
fragments. This is accomplished by the incubation
of the target DNA molecule with restriction
enzymes at the appropriate enzymatic conditions.
The restriction endonucleases are sequencespecific, so they inspect the length of DNA and trim
it at particular sites known as the restriction site.
Restriction digestion results in the production of
blunt ends (ends of a DNA molecule that ends with
a base pair) or sticky ends (ends of a DNA molecule
that end with a nucleotide overhanging). They are
called ‘sticky ends’ because they can be used to
rejoin fragments of DNA. They stick together by
forming hydrogen bonds to complementary sticky
ends from other DNA molecules (in this case the
plasmid or bacteriophage DNA)
2) CUTTING OF THE DNA/ RESTRICTION
ENZYME DIGESTION
The desired insulin gene and the vector is snipped by the
same restriction enzymes to acquire the complementary
sticky ends. This ensures the task of ligases for binding the
required gene to the vector is easier.
In insulin production, a bacterial plasmid is isolated and cut
open using restriction enzymes, creating a vector for the
insulin.
3) AMPLIFICATION USING PCR
Copies of genes are amplified through PCR or
polymerase chain reaction. It is essentially a process to
increase a single DNA copy into several copies after the
desired gene of interest is cut with restriction enzymes.
It allows a single copy or a few copies of DNA to be
amplified into thousands or millions of copies.
The following components are used in PCR reactions .
3) AMPLIFICATION USING PCR
The following components are used in PCR reactions
that are conducted on 'thermal cyclers':
Template: DNA that has to be amplified.
Primers: oligonucleotides are tiny, chemically produced
molecules that are complementary to a DNA region.
Enzyme: DNA polymerase.
Nucleotides: The enzyme is required to lengthen the
primers.
PCR can be used to amplify the cut DNA fragments,
which can subsequently be ligated with the cut vector.
Note, Polymerase Chain Reaction (PCR) is not typically
used in the production of insulin, as it is primarily used
to amplify specific DNA sequences.
4) LIGATION OF DNA MOLECULES/
JOINING OF DNA
The vector and a section of DNA are joined in this step.
It is achieved with the help of the enzyme DNA ligase.
With the same restriction enzyme, the pure DNA and
the vector of interest are cut.This yields the cut DNA
fragment and the cut vector, both of which are now
open.
Ligation is the process of putting these two parts
together with the enzyme 'DNA ligase.' The resulting
DNA molecule is a hybrid of the interest molecule and
the vector DNA molecules. Recombination is the term
used in genetics to describe the merging of different
DNA strands. As a result, this new hybrid DNA molecule
is known as a recombinant DNA molecule, and the
process is known as recombinant DNA technology.
In this step, the insulin gene and the plasmid vector are
ligated together using DNA ligase, forming a
recombinant plasmid.
5) INSERTING DNA INTO A HOST
In this stage, the process of adding rDNA to the
recipient host cell, which is most commonly a bacterial
cell, takes place and this procedure is termed
‘transformation’. Afterwards, the recombinant DNA
multiplies and manifests as manufactured protein
under favorable conditions. Additionally, as since
bacterial cells have a hard time accepting foreign DNA,
treatments to make them 'capable' of accepting new
DNA such as thermal shock, calcium ion therapy,
electroporation, and other procedures are
administered.
The recombinant plasmid containing the insulin gene is
introduced into host cells, such as bacteria or yeast,
through a process called transformation. The host cells
take up the recombinant plasmid and become
transformed.
5) INSERTING DNA INTO A HOST
6) ISOLATION OF RECOMBINANT CELLS
As a result of the insertion of the rDNA into a host
cell(transformation process), a mixed population of
converted and non-transformed host cells is formed
and during the stage of isolating the recombinant cell,
only the transformed host cells are filtered. In
distinguishing recombinant cells from nonrecombinant cells, the marker gene of the plasmid
vector/ selectable marker is used. By recombinant cell
isolation the cells having the gene of interest can be
grown on a small or large scale.
Transformed host cells containing the recombinant
plasmid are selected for and isolated. This is typically
done by using a selectable marker, such as antibiotic
resistance, that is included on the plasmid. The
transformed cells are then grown in large
fermentation tanks to produce insulin, which is
harvested and purified.
6) ISOLATION OF RECOMBINANT CELLS
Once a sufficient amount of insulin has been produced
by the bacteria, they are harvested .The insulin is then
extracted from the bacterial cells and purified to remove
any bacterial components and other impurities. Finally,
the purified insulin is formulated into the desired form
(e.g., solution, powder) and packaged for medical use.
Diagram Showing Insulin Production
More
Examples
GOLDEN RICE
1. Gene Cloning: The isolated genes are cloned, typically into a plasmid vector,
which serves as a carrier for the foreign genes.
2. Transformation of Rice Cells: The cloned genes are introduced into rice plant
cells using a method such as agrobacterium-mediated transformation or
biolistic bombardment (gene gun). This process can be performed in tissue
culture under controlled conditions.
3. Integration into Rice Genome: The introduced genes integrate into the rice
genome, often at random locations. This step is critical for stable inheritance of
the desired trait in subsequent generations of rice plants
4. Regeneration of Transgenic Rice Plants: The transformed rice cells are cultured
to regenerate whole plants. This involves selecting and nurturing cells that have
successfully incorporated the foreign genes and developed into viable plants.
Diagram Showing Golden Rice
Production
RECOMBINANT
VACCINNES
https://www.youtube.com/watch?v=DgDDw2gQJx0
QUESTIONS
1. Name the all six steps of Recombinant
DNA Technology in order
2. Give 3 examples of Recombinant DNA
Technology
Thank you!
REFERENCE
https://www.britannica.com/science/recombinant-DNAtechnology/Creating-the-clone (additional information)
https://www.youtube.com/watch?v=8cYvyYOjzOc (information
on DNA isolation)
https://www.vedantu.com/biology/recombinant-dnatechnology (information on the steps for recombinant DNA
technology)
https://www.genscript.com/what-is-restrictiondigestion.html#:~:text=Restriction%20digestion%20is%20acco
mplished%20by,outside%20of%20the%20recognition%20seque
nce. (information on restriction enzymes)
REFERENCE
https://www.evitria.com/journal/recombinantantibodies/recombinant-dna-technology/
https://www.nlm.nih.gov/exhibition/fromdnatobeer/exhibitioninteractive/recombinant-DNA/recombinant-dna-technologyalternative.html#:~:text=Recombinant%20DNA%20is%20a%20te
chnology,insulin%20gene%20in%20the%20laboratory.
(examples)
https://www.vedantu.com/biology/recombinant-dnatechnology (rDNA insertion into host, isolation of recombinant
cells)