Recombinant DNA technology involves joining DNA molecules from different sources and inserting them into a host organism to produce multiple copies. It was first done in 1973 by Boyer and Cohen. The process involves isolating DNA, cutting it with restriction enzymes, ligating the DNA pieces together, introducing the recombinant DNA into a host, and screening for the recombinant organisms. This technology is used to produce important proteins like human insulin in bacteria and create transgenic plants with useful traits such as pest resistance.

2. What is Recombinant DNA technology
• Recombinant DNA technology is a branch of
molecular biology deals with the joining of DNA
molecules from two different sources and
inserted into host organism to produce a number
of copies of the new genetic combinations which
find applicant in science , medicine , agriculture
and industry
• It was first conducted by Boyer and Cohen in 1973

3. Process of recombinant DNA technology
1
• Isolation of DNA
2
• Restriction digestion
3 • ligation
4
• Introduction of recombinant DNA into host
5
• Screening of recombinants
6
• Use for producing proteins and other applications

4. 1) Isolation of DNA
• DNA is isolated from the source of interest . DNA is
enclosed with in the membranes in the cell thus
one needs to digest the extra layer in order to
isolate the desired DNA , however cell does not
only contains DNA but other macromolecules too
hence it is necessary to extract DNA in pure form
• The required DNA whether from bacteria, animal
or plant cell can be achieved through treatment of
various enzymes in order to digest the membranes.
• RNA can be removed by treatment of ribonuclease
whereas proteins can be removed by the
treatment of protease.

5. • Enzymes such as lysozyme can be used to degrade bacterial cell
wall , chitinase to break fungal cell wall and Cellulase for plant
cell wall.
• Ultimately the DNA is precipitated under treatment of ethanol
and gives out foreheads these are then spooled out to give
purified DNA.

6. 2) Restriction enzyme digestion
• Discovered by Arber , Nathans and Smith .
• Restriction enzyme cut the DNA into specific nucleotide
sequence known as recognition or restriction site .
• The isolated DNA is cut into fragments by the help of
restriction enzymes , these restriction enzymes cut open the
fragments of isolated DNA as well as the vector DNA so the
fragment could be attached to it.

7. • A restriction enzyme either cut the DNA into blunt ends or
staggered /sticky ends
• Each restriction enzyme is specific to the particular sequence or
restriction site
• BLUNT ENDS : the cleavage occur exactly at the symmetry
• STAGGERED / STICKY ENDS : the cleavage is not on symmetrical
axis and thus the fragments have sticky or cohesive ends

8.  Isoschizomers : pairs of restriction enzymes the
recognize the same recognitions sequence and cut in
the same location
• For e.g. SphI (CGTAC/G) and BbuI (CGTAC/G)
Neoschizomers : recognize the same sequence but
cut it differently
• For e.g. SmaI (GGG/CCC) and XmaI(G/GGCCC)
Isocaudomers : recognize slightly different
sequences , but produces same ends
For e.g. Sau3A ( N/GATC) and BamHI(G/GATCC)
Sau3A has four base pair recognition sequence and
BamHI has six base pair recognition sequence

9. • Restriction enzymes cut the DNA fragment leaving
sticky ends on foreign DNA as well as on vector DNA ,
hence they could be attached to the other easily .

10. 3)Ligation
• DNA ligases join the molecules by synthesizing
phosphodiester bond between nucleotides at the ends of
two different molecules
• Here, the sticky end of the foreign DNA fragment base is pair
with complementary sticky ends of the vector DNA and
enzyme ligase catalyses the formation of bond hence joining
the two

11. Vectors
• Vectors acts as transporting vehicle which carries foreign
DNA into a host cell for the purpose of cloning and
expression.
• Important features for vectors are:
Ability to replicate in host cell i.e. origin of replication
Restriction enzyme sites for specific cleaving
Genetic marker to separate the transformed recombinant cell
• Different types of vectors :
 lambda phage vectors for viral based cloning
Bacterial plasmid is extrachromosomal circular DNA
Cosmid , hybrid of lambda phage and plasmid

12. Plasmid as vectors
• Plasmids are extrachromosomal DNA in bacteria, it can
replicate and thus can contribute in the function of the
cell.
• E.coli plasmids have been employed as vehicles since
long time for the transfer of specific gene
• All plasmid vectors have common features ;
A replicator
A selectable marker
A cloning site

13. • The origin of replication viz. ori is preset in the
plasmid
• plasmid must contain the selectable marker , it is
important for distinguishing between the transformed
and on transformed recombinant cell .
• For example ; if the plasmid contains the gene for
destroying the ampicilin , the host cell containing
ampicilin will sustain and will grow and the cells with
no ampicilin will be killed
• The cloning site is Endonuclease restriction site which
cleaves at the exact site so the foreign DNA could be
inserted

14. Typical plasmid vector
• This vector is pBR 322
• It has 4361 base pairs
• It comprises of HindIII ,
BamHI , EcoRI as the cleaving
Or cloning site
• Tetracycline (tet) and ampicilin
(amp) antibiotics marker
• Ori , the origin of replication

15. The nomenclature of the plasmid is done as;
• P (XY) (Numbers or ID)
• P is for plasmid
• (XY) is for initials of the discoverer
• (numbers or ID) is the lab isolated number or the name
• For example:
• pBR322
• P= plasmid
BR= viz. Bolivar and Rodriguez ,
 322= Is the lab isolate number

16. Introduction of recombinant vector
into the host
• The recombinant DNA is introduced
commonly in the bacterial cell
for the production of specific
protein and can be achieved by :
Chemical transformation
Electroporation

17. a)Chemical transformation
• If we take E.coli as the bacterial host cell in which the
recombinant DNA has to be introduced ,
• Ca ions are introduced in the cell medium, it reduces
the stability of the E.coli membrane
• Ca ions can induce the non- bilayer structures in
total lipids and can also enhance the phase transition
of phosphatidylglycerol and lipopolysaccharides
• DNA molecules are negatively charged hence are
repelled by the outer membrane, Ca+ ion neutralizes
and favours the transport

18. • Recombinant plasmid is incubated with host
cells on ice
• Subsequently brief heat shock allows the entry
of DNA through the adhesion zones

19. b) Electroporation
• It uses electric field to disturb the membrane of host
cell
• It allows the DNA to enter the host cell
• Advantages :
• It is effective with nearly all type of species
• Efficiency is high as large percentage if cells take the
DNA molecules
• Disadvantages :
• If electric pulse time is too long the pore could
became much larger leading to damage the cell
• The non specific material could enter the cell
resulting into ionic imbalance hence cell death

20. Screening of Recombinant DNA
• This allows us to distinguish between
the transformed and non transformed
host cells
• Common methods are:
Selection by alpha-complementation
Colony hybridisation
Polymerase chain reaction

21. Colony hybridisation
• A single strand of DNA from one organism is bound to a
nitrocellulose membrane ( binds single stranded DNA only )
• Radioactively labelled a single stranded DNA probe containing
DNA sequences of the foreign DNA is added to the
membrane .
• If the labelled DNA finds a complementary sequence , it will
bind it to and radioactive signal will be observed
• In this technique the bacterial colonies are grown on the agar
plate are transferred to nitrocellulose membranes , lysed
there, and the foreign DNA identified by hybridisation with a
suitably labelled probe.
• The colonies containing identical or similar DNA sequence will
produce signals , can be observed on the x ray film

22. α-Complementation / Blue – white
screening
• The bacterial cells are plated in to the medium
containing isopropyl thiogalactoside and the dye 5-
bromo-4-chloro-3-inodlyl-β-D-galactoside
(X-gel)
X-gel is colourless but on cleavage with enzyme, β-
galactoside yields a derivative which is blue in colour
• Plasmid containing lacZ gene will produce
β-galactoside
• this will give rise to blue colored colonies and rest will
produce white color
• Thus on the basis of color developed recombinant DNA
colonies can be separated

23. Polymerase chain reaction
• The cells are lysed and the DNA is inserted in pcr
• The primer comprises complementary DNA as of the
foreign DNA is added to pcr
• The primer hence will combine with the recombinant
DNA and will make copies of it
• This will separate the non recombinant from
recombinant one

24. Production of proteins
1) Recombinant technology in the synthesis of the
human insulin
• The first genetically engineered insulin was synthesized
in the laboratory of Herbert Boyer in 1977, usually
extracted from pancreas of cows and pigs. This insulin is
slightly different in structure from human insulin. As a
result, it leads to allergic reactions in about 5% patients.
Human gene for insulin production has been
incorporated into bacterial DNA and such genetically
engineered bacteria are used for large scale production
of insulin. This insulin does not cause allergy

25. 2)Development of Transgenic Plants:
• Genetically transformed plants which contain foreign
genes are called transgenic plants. Resistance to
diseases, insects and pests, herbicides, drought;
metal toxicity tolerance; induction of male sterility
for plant breeding purpose; and improvement of
quality can be achieved through this recombinant
DNA technology. BT-cotton, resistant to bollworms is
a glaring example.

26. Recombinant DNA technology