rDNA Technology

1. PADMASHREE DR.D.Y.PATIL COLLEGE
OF PHARMACY AKURDI
TOPIC NAME- RECOMBINANT DNA
TECHNOLOGY
SUBJECT NAME – CELLULAR AND MOLECULAR
PHARMACOLOGY
GUIDED BY- DR. PAWAN WANKHADE SIR
PRESENTED BY – DHANASHRI PRAKASH SONAVANE

2. INTRODUCTION:
• 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. The resulting
copies are often referred to as recombinant DNA.
• Ex: DNA Comprising animal gene may be recombined with DNA
From Bacteria.

3. Principle
• Every protein is encoded by a certain gene. If the gene can be
identified, then that can be used to produce a large quantity of the
same gene or product encoded by that gene. The gene of interest can
be transferred to a vector DNA to produce recombinant DNA or
cloned gene. Vectors are self-replicating inside an appropriate host
cell. Introduction of recombinant vectors into host cells cause
multiplication of the cloned gene. Expression of the cloned gene
produces desired protein.

4. Discovery Of Recombinant DNA
Technology
• Discovery of DNA Structure-Watson and Crick 1953.
• Isolation of DNA Ligase in 1967.
• rDNA Technology Developed by Paul Berg.
• Cohen and Bayer in 1973 produced 1st plasmid vector capable of
being replicated with bacterial host.

5. GOALS OF rDNA TECHNOLOGY
• To isolate and characterize gene.
• To make desired alteration in one or more isolated gene.
• To return altered gene to living cell.
• Artificially synthesize new gene.
• Alteration the genome of an organization.
• Understand heredity disease and their cure.
• Improve Human Genome.

6. Step of rDNA Technology
1. Isolation of desired DNA.
2. Isolation of vector.
3. Chimeric DNA production.
4. Introduction of the recombinant DNA into host cell.
5. Multiplication and selection of cell with DNA Expression of gene -
Product.
6. Separation and purification of the product.

7. 1. Isolation of Desired piece of DNA
In this DNA cell it
is surrounded by
cell wall.
Treat by enzymes,
detergent, protease
& ribonuclease.
Ultracentrifugation
is done and DNA is
obtain by, DNA
treatment with
chilled ethanol
Isolate DNA
These DNA
undergoes the
Restriction
Endonuclease
Separation of this
DNA fragment of
molecules with
sticky ends & Blunt
ends.
which get separated
by electrophoresis &
identified by southern
blotting technique.

9. 2. Isolation of vectors:
Plasmid (vector)
surrounded by
bacterial cell wall.
Treated by Enzyme,
EDTA, Lysozymes.
Then it is treated
with Sodium lauryl
sarcosinate.
It undergoes
Ultracentrifugation,
Electrophoresis sepa
ration You will get
plasmid .
Treat this vector
with restriction
endonuclease.
Then you will get
Plasmid with sticky
ends.

10. 3.Production/Hybridization of (Chimeric)
Base DNA
Formation of Hybridized Chimeric Base
DNA Ligase (DNA
joining enzyme) &
(it requires ATP de
formation of
Phosphodi -esterase
linkage)
Desired Piece
Of DNA
Plasmid
Molecule

11. 4. Introduction of the recombinant DNA
into host cell:

12. 5.Multiplication of Host cell to selection of
cell containing Chimeric DNA esters
• Done by Hybridization technique with the help of Probe Antibiotic Br by
using resistance.
6. Expression of gene to produce desired
product:
• After this desired piece of gene produce desired protein Process on
transcription & translation.
7. Separation and purification of the
product.

13. Study various tools used in rDNA
technology
• Enzymes
1. Exonuclease-cut outer nucleotide.
2. Endonuclease - cut act on internal Phosphodiesterase linkage.
A. Restriction endonuclease (also called as molecule seizure).
i. Restriction endonuclease 1
ii. Restriction endonuclease2
iii. Restriction endonuclease3
 1 and 2 required ATP they cut the DNA molecules away from
recognition sequence.
but 2 is important because it cut the DNA molecule within the
sequence.

14. • recognition sequence is 4-6 nucleotides and it 13 plandromic (rate
similar in either direction.
EX:
--G AA T T C--
--C T T AA G--
Single Strand of E.Coli Bacteria
They act on ;
--G AA T T C-- Then You Will Get --C T T AA G--
--C T T AA G-- -- G AA T T C--
Separated by Sticky And Blunt End.

15. B. DNA Ligase:
(DNA joining enzyme are also called molecular glue) require ATP &
Phosphodiesterase linkage Bond formation.
C. Polymerase
D. Alkaline Phosphate
E. Reverse Transcriptase

16. Vectors
• Vector is a DNA molecule in to which a gene is inserted to construct recombinant DNA molecule
and it is also capable of replicate in a host organism. It is also otherwise known as vehicle because
the vector transports the inserted gene to host organisms.

17. Gene Transfer Technique
• Transformation
• Transduction
• Electroporation
• Conjugation
• Direct Transfer of DNA
• Lipofection

18. Electroporation:
• It is based on the principle that high voltage electric pulses can induce
cell plasma membranes to fuse. It is a technique involving electric
field-mediated membrane permeation. Electric shock can also induce
cellular uptake of exogenous DNA (believed to be pores formed by
electric pulses) from the suspending solution.
Conjugation:
• In this method, two live bacteria (a donor and a recipient) come
together, join by cytoplasmic bridges and transfer single stranded
DNA from donor to recipient. Inside the recipient cell, the new DNA
may integrate with the chromosome (rare) or may remain free (as is
the case with Plasmids).

19. Transduction:
• Sometimes the foreign DNA can be packed inside animal viruses.
These viruses can naturally infect the cells and introduce the DNA into
host cells. The transfer of DNA by this approach is referred to as
transduction.
Direct Transfer of DNA :
• Microinjection and particle bombardment are the two techniques
commonly used to directly transfer the DNA into the cell nucleus.
Lipofection:
• The liposome mediated gene transfer is referred to as lipofection.
Liposomes are circular lipid molecules, which have an aqueous
interior that can carry nucleic acids.

20. Transformation
It is the method of introducing foreign DNA in bacteria cells ex: E.Coli
The uptake of plasmid DNA by E.Coli is carried out in:
• Ice-cold CaCl₂ (0-5°C).
• Ice-cold CaCl₂ affects the cell wall, breaks at localised regions and is
also responsible for binding of DNA to cell surface.
• A subsequent heat shock (37°C-45°C for about 90 seconds).
• A brief heat shock (e.g. the sudden increase in temperature from 5°C
to 40°C) stimulates DNA uptake.