Includes the basic structure of DNA and the outline of Recombinant DNA technology.

1. Recombinant DNA Technology
Presented by
Gayathri S Mohan

2. DNA- the genetic secret!!
Encodes the genetic
instructions of all known living
organisms and many viruses.
Nucleotides are the basic
building block.
 Nucleotide= Sugar +
phosphate + Nitrogen bases.
4 Nitrogen bases
Anti-parallel strands

3. Nitrogen bases
• Adenine (A)
• Guanine (G)
• Thymine (T)
• Cytosine( C)
found in pairs, with A & T and G & C Double helix
sequence and number of bases creates the diversity
DNA
mRNA
Proteins

4. What is Gene???
• A gene is a stretch of DNA
that codes for a type of
protein that has a function
in the organism.
• It is a unit of heredity in a
living organism.. All living
things depend on genes
• Genes hold the information
to build and maintain an

5. Recombinant DNA Technology
 Production of a unique DNA molecule by joining
together two or more DNA fragments not normally
associated with each other, which can replicate in
the living cell.
 Recombinant DNA is also called Chimeric DNA
 Developed by Boyer and Cohen in 1973
 3 different methods of DNA recombination
• Transformation
• Non-bacterial Transformation
• Phage induction

7. Recombinant DNA Technology
Basic steps involved in recombinant DNA technology
Isolation of the gene of interest
Preparation of Vector DNA and DNA to be cloned
Insertion of the gene to the vector molecule and ligation
Introduction of the vector DNA to the appropriate host
cell
Amplification of the recombinant DNA molecule in host
cell.

8. Overview of rDNA technology
Bacterial cell
Bacterial
chromosome
DNA containing
gene of interest
Plasmid
Isolate Plasmid
Gene of interest
Enzymatically cleave
DNA into fragments.
Isolate fragment with the
gene of interest.
Insert gene into plasmid.
Insert plasmid and gene
into bacterium.
Culture bacteria.

9. But let’s look at it in more
detail....

10. Isolation of gene
 DNA molecule is extracted from the cell by using cell lysing
method
Homogenization
Centrifugation
 Gene of interest is isolated using probes and electrophoresis
 DNA which is to be cloned have to be inserted in to a vector
molecule which act as a carrier of the DNA to the host cell.
 The choice of a vector depends on the design of the
experimental system and how the cloned gene will be
screened or utilized subsequently.
 Commonly used vectors are Plasmid, bacteriophage, cosmid,
bacterial artificial chromosome (BAC), yeast artificial
chromosome (YAC), yeast 2 micron plasmid, retrovirus,
baculovirus vector

11. Plasmid vector
Covalently closed, circular, double stranded DNA
molecules that occur naturally and replicate extra
chromosomally in bacteria and in some fungi.
Eg: pBR 322 and pUC-18
characteristic of an ideal plasmid
(antibiotic resistance gene, such as ampr and tetr
(i)Presence of minimum amount of its own DNA.
(ii) Recognition sites for restriction endonuclease
(iii)Presence of at least two markers with recognition
site being present in one of the two markers
(iv)Relaxed replication control so that the recombinant
plasmid is capable of forming several copies.
A plasmid containing resistance to an antibiotic (usually
ampicillin) or Tetracycline, is used as a vector.

12. Restriction Endonucleases
Important tool for rDNA technology is the Restriction Enzymes
 Bacterial enzymes that cut DNA molecules only at restriction sites
 Molecular scissors
 Palindromic sequences are the recognition sites
eg: EcoRI with recognition site GAATTC
5´ GAATTC 3´
3´ CTTAAG 5
 Categorized into two groups based on type of cut
• Cuts with sticky ends
• Cuts with blunt ends
if one strand extends beyond the complementary region, then the
DNA is said to possess an overhang and it will have sticky ends.

13. Commonly used restriction enzymes
•
•
•
•
•
•
•
•
EcoRI –
BamHI –
DpnI –
HindIII –
BglII –
PstI –
Sau3AI –
KpnI –
Escherichia coli strain R, 1st enzyme
Bacillus amyloliquefaciens strain H, 1st enzyme
Diplococcus pneumoniae, 1st enzyme
Haemophilus influenzae, strain D, 3rd enzyme
Bacillus globigii, 2nd enzyme
Providencia stuartii 164, 1st enzyme
Staphylococcus aureus strain 3A, 1st enzyme
Klebsiella pneumoniae, 1st enzyme

14. Restriction Endonucleases
Enzymes with staggered cuts  complementary ends
• HindIII - leaves 5´ overhangs (“sticky”)
5’ --AAGCTT-- 3’
3’ --TTCGAA-- 5’
5’ --A
AGCTT--3’
3’ –TTCGA
A--5’
• KpnI leaves 3´ overhangs (“sticky”)
5’--GGTACC-- 3’
3’--CCATGG-- 5’
5’ –GGTAC
C-- 3’
3’ –C
CATGG-- 5’
• Enzymes that cut at same position on both
strands leave “blunt” ends
SmaI
5’ --CCCGGG-- 3’
3’ --GGGCCC-- 5’
5’ --CCC GGG-- 3’
3’ --GGG CCC-- 5’

15. Actions of restriction enzymes-overview

16. Recombinant techniques
• DNA to be cloned and the vector molecule are
treated with the same restriction nuclease
separately
• It produces complimentary sticky ends
• Sticky ends will self ligate through covalent
bonding
• This results in recombinant DNA molecule

17. Ligation of DNA
DNA Ligases close nicks in the phosphodiester backbone of
DNA
 DNA ligase is a enzyme that can link together DNA strands
that have double-strand breaks (a break in both
complementary strands of DNA).
 Needs ATP
ATP

18. Cloning-Transformation
• It is introduced into host cell by adding it into
culture of plasmid free bacteria or animal
cells.
• Heating and adding calcium chloride favors
the transformation
• Once inside the host cell, the recombinant
DNA begins to multiply and form the desired
product.

19. Selection of recombinant cells

20. Selection of recombinant cells
• Only bacteria which
have taken up plasmid
grow on ampicillin.
• Blue-white selection:
– white colonies have
insert
– blue colonies have no
insert

21. Growing successfully….
• The transformed cell are cultured and multiplied.
• Colony of cell each containing the copy of the
recombinant plasmid is obtained.

22. Non-Bacterial transformation
Microinjection, using
micropipette.
The host cells are
bombarded with high
velocity micro-projectiles,
such as particles of gold or
tungsten that have been
coated with DNA.

23. Phage Introduction
• Phage is used instead of bacteria.
• In vitro packaging of a vector is used.
• lambda or MI3 phages to produce phage
plaques which contain recombinants.

24. Electroporation
• It involves applying a brief (milliseconds)
pulse high voltage electricity to create tiny
holes in the bacterial cell wall that allows
DNA to enter.

25. Applications…
 Pharmaceutical and Therapeutic Applications
Gene therapy
Medical diagnosis
Xenotransplants
 Agricultural Applications
Production of transgenic organisms

26. Environmental applications
• Many waste products of agriculture/industry do not break down
naturally/break down slowly.
Many bacteria have been GE capable of breaking down oil and other organic
wastes in Cheese making industry : GE Saccharomyces cerevisiae able to
dispose of whey by converting lactose to alcohol.
Agricultural waste products, eg. corn husks, contain cellulose that normally
decomposes slowly, can be converted into sugar by cellulase. Cellulase has
been inserted in E.coli making it useful in waste management/disposal
programs..

27. Any Questions??????...