RDNA TECHNOLOGY

PRESENTED BY:

SRISHTI PATEL
 1111111 Topic Slide No.

1. Definition 3

2. History 4

3. 3 Tools of Recombinant DNA 5-8

Technology

INDEX 4. 7 Steps of Recombinant DNA 9-17

Technology

5. Application of Recombinant DNA 18

Technology

6. Limitations of Recombinant DNA 19

Technology

7. Reference 20
 DEFINITION

A series of procedures that are used to join together (recombine) DNA segments. A recombinant DNA
molecule is constructed from segments of two or more different DNA molecules. Under certain
conditions, a recombinant DNA molecule can enter a cell and replicate there, either on its own or after
it has been integrated into a chromosome.
 HISTORY

• The possibility for recombinant DNA technology emerged with the discovery of restriction enzymes in 1968
by Swiss microbiologist Werner Arber oo, Hamilton 0. Smith, and Daniel Nathans ..
American microbiologist Hamilton 0. Smith purified type II restriction enzymes, which were found to be
essential to genetic engineering for their ability to cleave at a specific site within the DNA.
• American molecular biologist Daniel Nathans helped advance the technique of DNA recombination in 1970-
71 and demonstrated that type II enzymes could be useful in genetic studies.
• About the same time, American biochemist Paul Berg developed methods for splitting DNA molecules at
selected sites and attaching segments of the molecule to the DNA of a virus or plasmid, which could then
enter bacterial or animal cells.
• In 1973 American biochemists Stanley N. Cohen and Herbert W. Boyer became the first to insert
recombined genes into bacterial cells, which then reproduced.
3 TOOL OF RECOMBINANT DNA TECHNOLOGY

Restriction Host
Vectors
Enzymes Organism
 RESTRICTION ENZYMES

• These are enzymes which have restriction endonucleases that help in cutting, polymerases that aid in synthesis
of DNA and ligases that facilitate binding.
• The restriction endonucleases used in recombinant DNA technology have a vital role in outlining the location at
which the desired gene of interest is introduced into the vector genome.
• These are of two types usually, named as endonucleases and exonucleases. The endonucleases cut within the
strand of DNA whereas the exo nucleotides are involved in cutting the ends of the DNA.
• The restriction endonucleases are specific to palindromic sequences and cut the DNA at specific points . There
are 3 chief types of restriction endonuclease enzymes : Type-I Restriction Endonucleases, Type-II Restriction
Endonucleases and Type-Ill Restriction Endonucleases .
• They inspect the length of DNA and cut at the specific site known as the restriction site. This creates sticky ends
in the sequence.
• The gene of interest and the vectors are cut by the same restriction enzymes to acquire the corresponding
sticky ends, after which ligases help in binding the sticky ends.
 VECTORS

• The vectors help in carrying and integrating the desired gene. These form a very important part of
the tools of recombinant DNA technology as they are the ultimate vehicles that carry forward the
desired gene into the host organism.
• Plasmids and bacteriophages are the most common vectors in recombinant DNA technology that
are used as they have a very high copy number.
• The vectors are made up of an origin of replication- This is a sequence of nucleotide from where the
replication starts, a selectable marker - constitute genes which show resistance to certain
antibiotics like ampicillin; and cloning sites - the sites recognized by the restriction enzymes where
desired DNAs are inserted.
 HOST ORGANISM

• The organism in which the recombinant DNA is introduced is called the host organism.
• It is the ultimate tool into which the vector drives the gene of interest using the enzymes.
• Techniques like microinjection, gene gun, biolistic are employed to insert the recombinant DNA
within the organism.
• This can be also carried out using alternate heating and cooling or use of calcium ions.
7 STEPS OF RECOMBINANT DNA TECHNOLOGY

Isolation of Restriction Ligation of

Amplification
Genetic - 1➔ Enzyme - 3➔ DNA
Using PCR
Material Digestion Molecules

Insertion of Obtaining
Downstream
Recombinant Foreign Gene
Processing
DNA Into Host Product
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.
3 different restriction
enzyme digests of !\
plasmid DNA DIGESTION
\ \

• Restriction enzymes act as molecular scissors that cut DNA \t_ ,

specific locations. These reactions are called 'restriction enzyni'
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 l\
allows separating and cutting out the digested DNA fragments.
• The vector DNA is also processed using the same procedure.
 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 cyders' 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.
LIGATION OF DNA MOLECULES

CMV i"'""''°'
 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.
 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.
OBTAINING FOREIGN GENE
PRODUCT

• The recombinant DNA multiplies in the host and is expressed

as a protein, under optimal conditions. This is now a
recombinant protein.

• Small volumes of cell cultures will not yield a large amount of

recombinant protein. Therefore, large-scale production is
necessary to generate products that benefit humans. For this
purpose, vessels called bioreactors are used.

• Bioreactors are large containers with a continuous culture

system, where the fresh medium is added from one side and
used medium is taken out from another side.
 DOWNSTREAM PROCESSING

• Before the protein is marketed as a final product, it is subjected to downstream processing which
includes:
• Separation and purification .
• Formulation with suitable preservatives.
• Clinical trials to test the efficacy and safety of the product.
• Quality control tests.
APPLICATION OF RECOMBINANT DNA TECHNOLOGY
LIMITATIONS OF RECOMBINANT DNA TECHNOLOGY

Destruction of native
Resilient plants can II = ; . .Ill
species in the Cross contamination and
theoretically give rise to
environment the migration of proprietary
resilient weeds which can
genetically modified DNA between organisms.
be difficult to control.
species are introduced in.

Recombinant organisms A single disease or pest

Creation of superbug is
contaminating the natural can wipe out the entire
hypothesized.
environment. population quickly.
 REFERENCE

• Britannica - htt s: www.britannica.co recombinant-DNA-technolo

• Verma, P. S., & Agrawal, V. K. (2006). Cell Biology, Genetics, Molecular Biology, Evolution & Ecology
(1 ed .). S .Chand and company Ltd.
• Klug, W. S., & Cummings, M. R. (2003) . Concepts of genetics. Upper Saddle River, N.J: Prentice Hall.

recombinant-dna -technolo
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PRESENTED BY:

SRISHTI PATEL