RDT, rDNA, Biotechnology. upload for CBSE 12

1. BIOTECHNOLOGY
CLASS-XII
Recombinant DNA Technology-
Science uses various techniques to treat disease and improved their environment.
There are various techniques, out of which some techniques are used known as
Gene Cloning and RDT.
Previously this technique is used in production of human insulin (Diabetes).
For eg. Yeast cells- Hepatitis B vaccine
Plants such as Bt- Cotton (Insect resistant)
First time rDNA molecules generated by-Paul Berg, Herbert Boyer, Annie Chang
and Stanley Cohen in 1973.
These are important to produce large amount of rDNA (Gene cloning)
Large amount of protein expressed by insert.
These are important in today’s medical practice, essential to Pharma industry,
help to diagnose genetic disease such as Sickle cell anemia or used in
development of vaccine.
Recombinant DNA technology proved its importance in the production of vaccines
and protein therapies such as- Human insulin, interferon and human growth
hormone.
These are also used to produce clotting factor for treating Haemophilia and in the
development of gene therapy.
Application- In nutrition, in medicine to create pharmaceutical products (Human
Insulin).
# Importance of Plasmid-
These are relatively small DNA sequences that can self replicate and exist
independent of the chromosome.

2. Also carry antibiotic resistance genes that make them selectable.
Used to produce large number of copies at low cost.
Examples- Escherichia coli cells can be stored with the genes for human insulin in
their chromosomes.
 Pharmaceutical products.
 Vaccines
 Diagnostic testing
 Gene therapy
 DNA finger printing
 DNA and Agriculture
Types of Plasmid- there are various types of plasmids such as,
Fertility plasmid
Resistance plasmid
Virulence plasmid
Degradative plasmid Types
Col plasmid
Tools of rDNA Technology-
Restriction Enzymes :
The molecular scissors
Foundation of rDNA technology
Enzyme exist in many bacteria function as defense mechanism called restriction
modification system.
There are three types of restriction enzymes-
Type I, II, III
Out of which only Type II restriction enzyme is used in rDNA technology.
It recognize and cut DNA with in a specific sequence typically consisting of 4-8 bp.
Sequence referred to restriction site is generally Palindromic, both strand same.
Type II named on the basis of isolation of bacteria,
Eco RI
where E represents Genus,
co represents species,

3. R represents strain (RY 13)
I represents isolated for the first time
First discovered by – W, Arber, H.Smith and D.Nathans in 1978 and awarded by
Nobel prize
Table: types, microbial sources, recognition sequence- Ref. NCERT XII
Figure: Construction of rDNA using fragments from different sources,
Ref. NCERT XII.
Blunt end and Sticky end
DNA restriction sites cleaved by restriction enzymes results in
complementary ssDNA called Sticky ends.
Production of no complementary ssDNA results in blunt ends.
Restriction fragment length polymorphism (RFLP)-
 DNA of each organism has specific sequence which can be cleaved by
several restriction enzymes and fragments of different length can be
produced.
 These fragments are called restriction fragments.
 Restriction fragments of different individuals and species are different.
 These are of different length because of variation in DNA sequence of
restriction sites.
 These variation are called RFLP.
 Diagram
(Ref. NCERT XII)
Application of RFLP-
It is mostly used in DNA finger printing analysis technique.
Other enzymes used in rDNA Technology-
There are various enzymes that are used in rDNA technology

4. DNA Pol I -
a) It synthesizes DNA complementary to DNA template in 5’-3’
direction.
b) It lacks 5’-3’ exocatalytic activity.
Exonuclease III –
It cleaves from the end of a linear DNA and digest dsDNA from the 3’ end
only.
DNA ligases- Role in sealing ss nicks in ds DNA.
Forms phosphodiester bond between two adjacent nucleotide.
There are two types-
a) E.coli DNA ligase- Uses NAD as a source of energy.
b) T4 DNA ligase- Uses ATP.
Alkaline Phosphate (AP)-
a) 5’ phosphate group is essentially required for ligation of DNA fragments.
b) DNA can’t be ligated if 5’- phosphate group is removed.
c) The enzyme removes 5’ Phosphate group from 5’ DNA fragments and make
it free.
d) Enzyme is used for self-ligation.
There are different sources, Bacteria (BAP) and Calf Intestine (CAP).
Reverse Transcriptase-
RNA dependent DNA polymerase synthesizes DNA complementary to a RNA
template in 5’-3’ direction.
RNase A-
It is a nuclease which digest RNA but not DNA.
Tag DNA Polymerase-
DNA Pol isolated from a thermophilic bacteria (Thermus aquaticus)
Operates at 72 degee celcius.
Stable above at 90 degree celcius.
Used in PCR.
Terminal Transferase-
Enzyme adds several nucleotides to 3’ end of a linear ds DNA or RNA.

5. SI Nuclease-
SI nuclease acts on single strands of double stranded DNA and result blunt ends in
DNA fragments.
Vectors-
These are the vehicle used for cloning.
For eg. Plasmid
Features of vectors-
Ori (independently replicate with in host).
Incorporate a selectable marker.
Identify host cell containing vector.
Selectable Marker-
It include gene conferring antibiotic resistance.
Enzyme such as beta-galactosidase.
Gene expressing green fluorescent protein (GFP).
Easy to viewed under UV light.
Vector must have one unique restriction enzyme recognition site, used for
cutting and introducing an insert.
More than onr restriction site.
Contain a multiple cloning site (MCS) or Polylinker.
Provides flexibility in the choice.
It should be small in size so that entry into host cells.
Features-
It should easily be isolated from the organisms.
It should be small,
Larger vector DNA get broken during purification.
Origin of replication.
Contain a multiple cloning site.
Selectable marker (Helps to select the host cells from population).
Examples of Common selectable markers-
Genes providing antibiotic resistance

6. Eg. Ampicillin (ampr
), tetracyclin (tetr
), kanamycin (Kanr
).
Beta-galactosidase (Presence of this enzyme is identified by the change in color of
substrate).
Cloning vector Genome size (kb) Insert size (kb)
Bacteriophage lamda 48.514 9-23
Cosmids 30-45
pBAC 300-500
pYAC 1,000-2,500
Plasmid 1-1.50 0.5-8
M13 6.407 Less than 5
Plasmids-
Plasmids are extra-chromosomal
Self replicating
Circular
ds DNA molecules
Found in bacteria and yeast
Plasmids are not essential for normal cell growth & division.
Earliest plasmid vector
pBR322
contain two different antibiotic resistance genes recognition sites for several restriction enzymes
Shuttle vector-
Plasmid vectors can replicate only in Escherichia coli.
Many of them are used in eukaryotic cells.
They can exist in both.
In eukaryotic and in Escherichia coli such vectors are known as shuttle vectors.
Example of shuttle vector-
Yeast Plasmid Yep,
Naturally occurring plasmid of the bacterium ,
Agrobacterium tumefaciens called Ti plasmid.

7. Vectors based on bacteriophages-
Bacteriophages are viruses that infect bacterial cells by infecting their DNA
into them.
Take over the machinery of bacterial cells to multiply themselves.
DNA is selectively replicated and expressed in the host bacterial cell.
Resulting in number of phages out of the cell by lytic pathway .
Infect neighbouring cells.
Ability to transfer DNA from the phage genome to specific bacterial host during
the process of viral infection.
Two phages that have been extensively modified for the development of cloning
vectors are-
a. Lamda
b. M13 phages