RECOMBINANT DNA TECHNOLOGY
Recombinant DNA (rDNA) technology is a powerful scientific method used to
modify the genetic material of organisms. It involves combining DNA from two
different sources to create new genetic combinations that are not naturally found
in organisms. This is achieved by cutting DNA molecules using special enzymes
called restriction enzymes, inserting the desired gene into a vector (like a plasmid),
and introducing it into a host of an organism
RECOMBINANT DNA TECHNOLOGY
HOW RECOMBINANT DNA TECHNOLOGY WORKS?
Recombinant DNA technology involves several steps in
specific sequence such as
1. Isolation of DNA
2. Fragmentation of DNA by restriction endonucleases
3. Isolation of desired DNA fragment
4. Ligation of DNA fragment into suitable vector
5. Transferring the recombinant DNA into the host
6. Culturing the host cells in a medium at large scale
7. Extraction of the desired product
RECOMBINANT DNA TECHNOLOGY
ISOLOATION OF DNA
STEPS INVOLVED IN ISOLATION OF DNA:
1. Breaking the Cell (Cell Lysis):
o Cells containing the genetic material are broken open to release their
contents.
o This is done using enzymes:
▪ Lysozyme for bacterial cells.
▪ Cellulase for plant cells.
▪ Chitinase for fungal cells.
2. Removal of Other Cellular Components:
o After breaking the cells, other macromolecules like proteins, lipids, and
RNA are removed:
▪ Proteins are removed by treatment with proteases.
▪ RNA is removed by treatment with ribonuclease (RNase).
3. Purification of DNA:
o The DNA is then purified by using ethanol precipitation:
▪ DNA is precipitated out as a fine thread-like structure after the
addition of chilled ethanol.
▪ The DNA can then be spooled out or collected for further use.
The final result is purified DNA, which can be used for cutting with restriction
enzymes in the next step of recombinant DNA technology.
RECOMBINANT DNA TECHNOLOGY
FRAGMENTATION OF DNA BY RESTRICTION ENDONUCLEASES
1. Restriction Enzymes:
• Restriction enzymes are molecular scissors that cut DNA at specific sequences
called recognition sites (Hind II cuts DNA at specific sequence of 6 base
pairs)
• For example, the enzyme EcoRI recognizes the sequence GAATTC and cuts
between G and A.
2. Sticky Ends and Blunt Ends:
• When DNA is cut, it produces:
o Sticky ends: Overhanging single-stranded sequences that can easily
join with complementary sequences.
o Blunt ends: Straight cuts with no overhangs.
• Sticky ends are more commonly used in recombinant DNA technology as they
help in easy ligation of DNA fragments.
3.Fragmentation:
• The DNA is cut into fragments using restriction enzymes at specific
recognition sites.
• The enzyme cuts the DNA at specific sites, generating fragments of different
sizes.
RECOMBINANT DNA TECHNOLOGY
RECOMBINANT DNA TECHNOLOGY
ISOLATION OF DESIRED DNA FRAGMENTS
After cutting DNA with restriction enzymes, the fragments are separated using
agarose gel electrophoresis, where DNA moves based on size under an electric field.
The desired fragment is visualized with a dye (like ethidium bromide) under UV
light, cut out from the gel, and purified using elution methods. This isolated fragment
is then ready for further steps in recombinant DNA technology.
RECOMBINANT DNA TECHNOLOGY
LIGATION OF DNA FRAGMENT INTO A VECTOR
The desired DNA fragment is inserted into a plasmid vector using the enzyme
DNA ligase, which seals the bond between the fragment and the vector's
sticky or blunt ends. This creates a recombinant DNA molecule, ready for
transformation into a host cell.
RECOMBINANT DNA TECHNOLOGY
TRANSFERRING THE RECOMBINANT DNA INTO HOST
After ligating the DNA fragment into a vector (like a plasmid), the recombinant DNA
is introduced into a host cell using different methods tailored to the type of
organism. Here’s how the process works for both:
1. In Animals (e.g., Mammalian Cells):
• Microinjection: The recombinant DNA is directly injected into the nucleus of
a target animal cell using a fine needle.
• Electroporation: The host cells are exposed to an electrical pulse, which
creates temporary pores in the cell membrane, allowing recombinant DNA to
enter.
• Viral Vectors: Modified viruses are used to deliver the recombinant DNA into
animal cells. The virus infects the cell, transferring the gene into the genome.
2. In Plants:
• Agrobacterium-mediated Transformation: The bacterium Agrobacterium
tumefaciens, which naturally transfers DNA to plants, is used as a vector. The
recombinant DNA is inserted into the bacterium, which then transfers it into
plant cells.
• Gene Gun (Biolistic Method): In this method, tiny gold or tungsten particles
coated with recombinant DNA are shot into plant cells using high-pressure
gas, allowing the DNA to enter.
RECOMBINANT DNA TECHNOLOGY
RECOMBINANT DNA TECHNOLOGY
RECOMBINANT DNA TECHNOLOGY
CULTURING THE HOST CELLS IN A MEDIUM AT LARGE SCALE
culturing host cells in a large medium refers to growing a large number of cells in a
controlled environment to produce sufficient amounts of recombinant proteins or
other products. This is typically done in bioreactors, where conditions like
temperature, pH, and nutrient levels are carefully monitored to optimize cell growth
and productivity.
For animal cells, this process involves the use of nutrient-rich media to support cell
division and expression of the recombinant genes. For plant cells, similar culturing
methods are used, often combined with growth hormones to induce differentiation.
This process is critical for scaling up the production of therapeutic proteins,
enzymes, or even vaccines in biotechnology. The goal is to provide the right
conditions for host cells to grow and express the desired product in large quantities.
RECOMBINANT DNA TECHNOLOGY
EXTRACTRION OF DESIRED PRODUCT
the extraction of a desired product involves cloning a gene of interest into a
suitable vector, expressing it in a host organism, and then isolating the
product through processes like cell disruption, separation, and purification.
This approach allows for the large-scale production of valuable bioproducts
such as proteins, enzymes, or hormones, which can be used in medicine,
industry, and research. The process is crucial for the efficient and cost-
effective production of biologically derived products.
RECOMBINANT DNA TECHNOLOGY
CCONCLUSION
In conclusion, rDNA (recombinant DNA) technology has
revolutionized biotechnology by enabling the manipulation and
combination of genetic material from different organisms. This
technology allows for the production of genetically modified
organisms (GMOs) with desirable traits, the synthesis of important
bioproducts like insulin, vaccines, and enzymes, and advancements in
fields such as medicine, agriculture, and industry. By enabling precise
gene editing and protein production, rDNA technology has contributed
to significant advancements in healthcare, agriculture, and
environmental management, offering solutions to many global
challenges.
