Process of rDNA Technology, various vectors and restriction endonucleases used in rDNA along with its applications.

1. Recombinant DNA
Technology
Presented to: Dr. Nirmal Singh
Presented by: Ramanpreet Kaur
Class: M.pharmacology Sem-1
Roll no.: 22301102

2. Contents
• Basic principles of Recombinant DNA Technology
• Restriction enzymes
• Various types of vectors
• Applications of Recombinant DNA Technology

3. What is DNA?
• DNA= DeoxyriboNucleicAcid
• DNA is a polymer of Deoxyribonucleotides.
• A nucleotide is composed of three
components-
1. Phosphate group
2. Five carbon sugar- Deoxyribose
3. Nitorgenous Base- Adenine, Guanine, Thymine, Cytosine
• Represents Genetic Material of organism.

4. Recombinant DNA Technology
•Recombinant DNA technology is
the joining together of DNA
molecules from two different
species to produce new genetic
combinations that are of value to
science, medicine, agriculture
and industry.
•e.g.DNA comprising animal gene
may be recombined with DNA
from bacterium

5. Discovery of rDNA Technology
• Discovery of DNA Structure – Watson and Crick in
1953
• Isolation of DNA ligase in 1967
• Isolation of REase in 1970
• First Recombinant DNA was constructed by Cohen
and Boyer in 1972

6. Basic process of rDNA Technology
1. Isolation of foreign DNA- The first and initial step in Recombinant DNA
technology is to isolate the desired DNA in its pure form.
From where we get gene of interest
• Genomic library
• cDNA library
• Chemical synthesis of gene( sequence is known)
2. Selection of cloning vector- Vectors used as vehicle to artificial carry a
foreign DNA into another cell where it can be replicated.
Vectors include plasmids, bacteriophages, cosmids and artificial
chromosomes.

7. 3. DNA Cutting- Target DNA and vector are cut, so that two different
DNA molecules can be joined to form Recombinant DNA.
• DNA cutting may be done by restriction endonuclease enzyme.
4. Joining two DNA segments-With the help of DNA ligase enzyme
sticky ends of two DNA are joined.
5. Transformation- Transfer Recombinant DNA into host cell.
Mostly, Host cell is bacteria.
Physical methods Chemical metohds
✓Electroporation ✓PEG mediated
✓Microinjection ✓Cold CaCl2 method
✓Liposome mediated transfer

8. 6. Selection of tansformed Host cell-
After transformation, three different possibilities arise:
• No transformation takes place
• Transformation takes place however plasmid enters the host cell
• Transformation takes place and recombinant DNA enter the cell.
These types of cells are selected.
✓Anti antibiotic resistance in a selective medium
✓Visual characters
✓Assay for biological activity
✓Colony hybridization
✓Blotting test
7. Multiplication of introduced DNA in the host

10. Ligase
• Ligase is an enzyme which forms sugar
phosphate bonds between ends of DNA
fragments cleaved by restriction
endonucleuses.
• Molecular glues.
• It requires ATP and Mg2+ as co factors
for executing ligase action.
• Most common source of ligase is
bacteria.
• Most common ligase in a Recombinant
DNA technology is T4 DNA Ligase
• It is isolated from E.coli which is infected
with bacteriophage T4.

11. Vectors used in rDNA Technology
• Vectors are DNA molecules
that can carry a foreign DNA
segment and replicate inside
host cell.
• Also called Vehicle DNA or
Carrier of DNA.

12. DNA vectors
TYPES
1. PLASMIDS
pBR322 Vector
pUC Vectors
FEATURES
• Extra chromosomal
• Self replicating
• Usually circular
• Double stranded
• pBR322 is ideal plasmid vector
• Others are- pBR325, pBR327, pBR328
• p- plasmid
• BR- Boliver and Rodriguez who constructed this
plasmid
• 322-Number given
• P-plasmid ,UC- University of California
• More efficient and replaces pBR vectors ( size smaller,
Copy no. Of pUC is high)

14. TYPES
2.Bacteriophage
• Lambda phage
vector
• M13 phage vector
FEATURES
• Viruses that infect bacteria.
• A lambda phage is a bacteriophage that
infects E.Coli.
• Ends are sticky and referred as cos sites.
• Double stranded DNA of 48,502 bp.
• Filametnous phage.
• Single stranded DNA of 6407 bp.

16. TYPES
• Cosmid
FEATURES
• Combination of COS+ MID
• COS - COS site of Lambda phage
• MID – Plasmid DNA
• Properties of both plasmids and
bacteriophages.
• Clone DNA fragments upto 45 kb
• Used to make gene libraries

17. Types
• PHAGEMID
• Artificial Chromosomes
• Bacterial Artificial
Chromosomes (BAC)
• Yeast Artificial
Chromosomes
FEATURES
• Derived from:
✓Phage from M13
✓Mid from Plasmid
• Used for carrying larger DNA sequences.
• Artificial chromosomes are cloning vectors
that can carry DNA inserts orders of
magnitude larger than is possible with
plasmids or Lambda phase derived vectors.
• Accomodate upto 300-350 kb of foreign DNA
• Used in genome sequencing project.
• Clone DNA fragments of more than 1 Mb.
• Used extensively in mapping the larger
genomes i.e. in the Human Genome Project

19. Transpsons as Vectors:
• Transposons are units of DNA.
• Mobile (move from one DNA molecule to another)
Shuttle Vectors:
• Exist in both Eukaryotic cell and E.coli.
• Contain two types of origin of replication and selectable marker genes, one
type that functions in eukaryotic cell and another that functions in E.coli.
• Most of Eukaryotic vectors are shuttle vectors.
• e.g. Yeast episomal plasmid Yep.

20. Desirable properties of Vector
• Origin of replication
• Selectable Markers
• Cloning sites (Recognition Sites)
• Optimal size
• Self replicating

21. Restriction Endonucleases
• Also called ‘molecular scissors or biological scisoors’.
• Isolatesd for first time by W.Arber in 1962 in bacteria.
• Cut DNA duplex at specific points, result in formation of same set of
DNA fragment.
• Isolated from bacteria.

22. Types of Restriction Endonucleases
Type | Type || Type |||
Enzyme structure consists of three
different sub units.
Enzyme structure is simple. Enzyme structure consists of two
different sub units.
They require ATP, Mg2+, S-
adenosyl-methionine for
restriction.
They require Mg2+for restriction. They require ATP, Mg2+, S-
adenosyl-methionine for
restriction.
They recognize specific sites within
DNA but do not cut the sites.
They recognize specific sites within
DNA and cut these sites.
They recognize specific sites within
DNA but do not cut these sites.
They are not used in Recombinant
DNA technology.
They are used in Recombinant DNA
technology.
They are not used in Recombinant
DNA technology.
Note: Only restriction enzyme type II are used in gene manipulation because:
✓No ATP is needed for the cleaving action.
✓It makes cut in both the stands of DNA molecule.

23. Nomenclature of Restriction Endonucleases
• Restriction enzymes are named for the bacterium from which they have been
isolated.
✓ The 1st letter = 1st letter of the bacterium’s genus name ( in Italics)
✓The 2nd and 3rd letter = species (also in Italics)
✓The 4th letter = 1st letter of the strain. It is written in capital.
✓The end of the name indicates the order in which the enzyme was isolated. It is
written in Roman number.
e.g. The Enzyme Eco R1 is named as follows:

24. Restriction Endonucleases produce two types of cuts:
Sticky ends
• Sticky ends produce an
overhang.
• Easy to ligate.
• Two types of extensions may be
produced:
(a) 5’phosphate extension
(b) 3’ hydroxyl extension
• Blunt ends
• Blunt ends are produced when
they cut the DNA at the centre
of the recognition sequence.
• Difficult to join.
• High concentration of ligases,
linkers, adaptors are required.

26. Palindromic Nucleotide Sequence
• Special sequence in the DNA recognised by restriction
endonuclease is called palindromic nucleotide
sequence.
• The palindromes in DNA are base pair sequences that
are same when read forward (left to right) or backward
(right to left) from a central axis of symmetry.

27. Examples of Restriction Endonucleases

28. Applications of Recombinant DNA Technology
Agriculture
To produce genetically-
modified organisms such
as:
• Flavr Savr tomatoes
• golden rice rich in
proteins
• Bt-cotton to protect
the plant against ball
worms and a lot more.
Medicines
• Recombinant
DNA technology
is used for the
production of
Insulin.
• Vaccine
production
• Antibodies

29. Gene therapy
It is used as an
attempt to
correct the gene
defects which
give rise to
heredity diseases.
Diagnosis
• Detect the
presence of HIV
in a person.
• ELISA

30. Energy
applications
• Biohydrogen
• Bioethanol
• Biomethanol
• Biobutanol
• Xenotranspl
antation
• Cell/ tissue
culture