RECOMBINANT DNA

TECHNOLOGY
PRESENTED BY :- URWELA
SAHARE
INTRODUCTION
• Recombinant DNA technology refers to the joining together
of DNA molecules from two different species that are inserted
into a host organism to produce new genetic combinations that
are of value to science, medicine, agriculture, and industry.
• Recombinant DNA (rDNA), on the other hand is the general name
for a piece of DNA that has been created by the combination of at
least two strands.
• They are DNA molecules formed by laboratory methods of genetic
recombination (such as molecular cloning) to bring together
genetic material from multiple sources, creating sequences that
would not otherwise be found in the genome.
• 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
Steps of Genetic Recombinant
technology
Isolation of Genetic Material
Restriction Enzyme Digestion
Amplification Using PCR
Ligation of DNA Molecules
Insertion of Recombinant DNA Into Host
Isolation of Recombinant Cells
1. Isolation of Genetic Material

• The first step in rDNA 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.
2. Restriction Enzyme Digestion

• Restriction enzymes act as molecular scissors that cut DNA

at specific locations. 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 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.
3. Amplification Using PCR
• 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:
Template – DNA to be amplified
Primers – small, chemically synthesized oligonucleotides that
are complementary to a region of the DNA.
Enzyme – DNA polymerase
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
4. Ligation of DNA Molecules

• 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 cut vector,
that is now open.
• 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
5. Insertion of Recombinant DNA Into Host

• In this step, the recombinant DNA is

introduced into a recipient host cell
mostly, a bacterial cell. This process
is ‘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.
6.Isolation of Recombinant Cells

• The transformation process generates a mixed

population of transformed and non-trans- formed host
cells.
• The selection process involves filtering the transformed
host cells only.
• For isolation of recombinant cell from non-recombinant
cell, marker gene of plasmid vector is employed.
• For examples, PBR322 plasmid vector contains different
marker gene (Ampicillin resistant gene and Tetracycline
resistant gene. When pst1 RE is used it knock out
Ampicillin resistant gene from the plasmid, so that the
recombinant cell become sensitive to Ampicillin.
Tools of Recombinant DNA
Technology1
1.Restriction Enzymes - Cut DNA at specific sequences
Types: Type I, Type II (most common), Type III.
2. Vectors- DNA carriers for gene transfer.
Examples:Plasmids (circular DNA).Bacteriophages (virus-
based).Cosmids, YACs, BACs (large DNA fragments).
3. DNA Ligase - Joins DNA fragments by forming
phosphodiester bonds.
Example: T4 DNA Ligase.
4. Host Cells - Environment for DNA replication and
expression.
Examples:E. coli (prokaryotic).Yeast (eukaryotic).Mammalian
cells (complex protein production).
5. PCR and Gel Electrophoresis
VECTORS
• A vector is a substance, usually a piece of DNA that carries a
sequence of DNA or other genetic material and introduces it
into a new cell.
• Vectors act as vehicles to transfer genetic material from one cell to
the other for different purposes like multiplying, expressing, or
isolation.
• Vectors are used as a tool in molecular cloning procedures so as to
introduce the desired DNA insert into a host cell.
• The DNA insert that is transmitted by a vector is termed
recombinant DNA, and the process is also known as
recombinant DNA technology.
• Usually, the vectors are DNA sequences that carry different parts
involved in different functions. Vectors usually have an insert, also
known as a transgene, that carries the recombinant DNA and a
larger sequence called the backbone of the vector responsible for
the structure of the vector.
TYPES OF VECTORS
• 1. Plasmid Vectors :- Circular, double-stranded
DNA molecules found in bacteria.Commonly used
for cloning small DNA fragments.
Examples: pBR322, pUC series.
• 2. Bacteriophage Vectors :- Derived from viruses
that infect bacteria, such as λ phage.Capable of
carrying larger DNA fragments compared to
plasmids.
• 3. Cosmids :- Hybrid vectors combining features of
plasmids and bacteriophages.Carry large DNA
fragments (up to 45 kb).Require a helper phage for
packaging DNA
• 4. Yeast Artificial Chromosomes (YACs) :- Used for
cloning very large DNA fragments (up to 1 Mb).Contain
elements necessary for replication in yeast cells, such
as centromeres and telomeres.
• 5. Bacterial Artificial Chromosomes (BACs) :- Based on
the F-plasmid of E. coli.Used for cloning large DNA
fragments (up to 300 kb).Often employed in genome
sequencing projects.
• 6. Viral Vectors :- Modified viruses used to deliver
genetic material into host cells.
Common types:
Retrovirus vectors : Integrate genetic material into the
host genome.
Adenovirus vectors: Non-integrating, suitable for
transient expression.
Lentivirus vectors: Effective for both dividing and non-
dividing cells
• 7. Phagemid Vectors :- Hybrid vectors
combining features of plasmids and
bacteriophages.Require helper phages for
replication and packaging.
• 8. Ti Plasmid Vectors :-Derived from
Agrobacterium tumefaciens.Commonly
used for genetic modification of plants.
APPLICATION
• 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.
• Recombinant DNA is used to identify, map and sequence
genes, and to determine their function.
• Recombinant proteins are widely used as reagents in
laboratory experiments and to generate antibody probes
for examining protein synthesis within cells and organisms.
• Many additional practical applications of recombinant DNA
are found in industry, food production, human and
veterinary medicine, agriculture, and bioengineering.
LIMITATION
• Destruction of native species in the environment the genetically modified
species are introduced in.
• Resilient plants can theoretically give rise to resilient weeds which can be
difficult to control.
• Cross contamination and migration of proprietary DNA between organisms.
• Recombinant organisms contaminating the natural environment.
• The recombinant organisms are population of clones, vulnerable in exact same
ways. A single disease or pest can wipe out the entire population quickly.
• Creation of superbug is hypothesized.
• Ethical concern about humans trying to play God and mess with the nature’s
way of selection. It is exaggerated by the fear of unknown of what all can be
created using the technology and how is it going to impact the civilization.
• Such a system might lead to people having their genetic information stolen and
used without permission.
• Many people worry about the safety of modifying food and medicines using
recombinant DNA technology.