Biotechnology refers to the use of living organisms or their components to develop products or perform processes for specific use. Recombinant DNA technology uses genetic engineering techniques to create recombinant DNA by cutting and joining DNA molecules containing different genetic material. This allows genes to be transferred between organisms for applications such as producing human insulin or diagnosing diseases. The key steps involve isolating DNA, using restriction enzymes to cut the DNA at specific sites, joining DNA fragments using DNA ligase, and inserting the recombinant DNA into a host cell where it can be replicated through bacterial transformation.

1. Prepared by:
Mrs. Priyanka Tyagi
PGT (Biology)
K.V. Dogra Lines Meerut
Cantt
Biotechnology :
Principles and
Processes

2. What is biotechnology?
Biotechnology refers to the
technology using biology, which
has applications in agriculture,
food processing industry,
medicine diagnostics,
bioremediation, waste treatment,
and energy production.

3. Biotechnology
 Biotechnology deals with the techniques of
using live organisms or enzymes from organisms
to make products and processes that benefit
human beings.
 The definition given by European Federation of
Biotechnology (EFB) is as follows:
“The integration of natural science and
organisms, cells, parts thereof, and molecular
analogues for products and services.”

4. Principles of Biotechnology
 Genetic engineering : Introduction of
foreign genetic material (DNA/RNA)
into the host’s genome and altering its
phenotype.
 Aseptic techniques : Maintenance of
sterile atmosphere to enable growth
of only the desired cell in large
quantities for the manufacture of
products like antibiotics, vaccines
enzymes etc.

5. Techniques Of Genetic
Engineering
Creation of recombination DNA
Gene transfer into host organism
Gene cloning

6. Creation Of Recombination
DNA
 Stanley Cohen and Herbert Boyer
(1972) constructed the first
recombination DNA.
 They isolated the antibiotic resistance
gene from the Plasmid of the
bacterium Salmonella typhimurium.
 This piece of DNA carrying antibiotic
resistance gene was cut at specific
location by restriction endonuclease,
popularly known as Molecular Scissors.

7. Gene transfer
 The cut piece of DNA was
introduced in the plasmid of
Escherichia coli which acted as the
vector.
 The piece of DNA was ligated to
the vector plasmid by DNA ligase.
 This joining of the two DNA pieces
resulted in the creation of
recombinant DNA.

8. Gene cloning
 The new recombinant DNA was
transferred into E. coli.
 The r DNA replicated autonomously
by using the host DNA polymerase
enzyme and made multiple copies.
 The ability to multiply copies of any
template of DNA is called gene
cloning.

9. Tools of Recombinant
DNA Technology
Restriction enzymes
Cloning Vector
DNA polymerase enzyme
DNA Ligase enzyme
Host organism

10. Restriction enzymes
As
biological
scissors
!!!!

11. Restriction enzymes
 Restriction enzmes belong to a class
of enzymes called nucleases.
 These are of two types –
exonucleases and endonucleases.
 Exonucleases cut the DNA at the
ends.
 Endonucleases make cuts at specific
points in the DNA.

12. Restriction enzymes
 The first restriction endonuclease
isolated – Hind II.
 It was isolated from the bacterium
Haemophilus influenzae.
 Today we know more than 900
restriction enzymes isolated from
230 strains of bacteria.

13. Recognition sequence
 Restriction enzymes always cut DNA
molecules at a particular point by
recognizing a specific sequence of
base pairs.
 This specific base sequence is known
as recognition sequence.
 Example: EcoRI recognises only the
following sequence.
5’-------GAATTC-------3’
3’-------CTTAAG-------5’

14. Restriction Enzymes…….How do you
denote them????
• Names use 3-letter italicized code:
 1st letter - genus
 2nd & 3rd - species
• Following letter denotes strain
EcoRI
was the first restriction enzyme found in the R strain of E. coli

15. Palindromic Sequence
 Palindromes are those group of
letters which read the same, both
forward and backward. Ex.
MALYALAM
 A Palindromic sequence is a sequence
which reads the same on the two
strands of DNA when orientation is
kept the same.
5’-------GAATTC-------3’
3’-------CTTAAG-------5’

16. Cutting & Pasting…….Restriction enzymes as
molecular scissors
 Restriction enzymes
◦ Three types: Types I, II and III
◦ Type II used for molecular biology work
◦ Endonucleases
◦ Recognize specific sequence of base-pairs,
usually 4, 6 or 8 bases that are palindromic
◦ Can leave ‘sticky ends’ or ‘blunt ends’

17. 5’-ATGCGAT-3’
3’-TACGCTA-5’
EcoRV
5’-ATGCG-3’
3’-TACGCTTAA-5’
EcoR1
5’ ATGCGAATTCCGGAA 3’
3’ TACGCTTAAGGCCTT 5’
5’ ATGCGATATCCGGAA 3’
3’ TACGCTATAGGCCTT 5’
Sticky ends
Blunt ends
5’-AATTCCGGAA-3’
3’-GGCCTT-5’
5’-ATCCGGAA-3’
3’-TAGGCCTT-5’
About sticky and blunt ends………

21. What do restriction enzymes
help us achieve ???
• They help in generating DNA fragments with precise ends
• These precisely generated ends can then be ‘pasted’ to similar
ends in a vector molecule that is self replicating.
• The process of ‘pasting’ is called ligation and requires a DNA
ligase
• The product after ligation is called a ‘recombinant DNA
molecule’ or a ‘clone’
• Each clone can be replicated to provide ample material for study

22. Cloning vector is a DNA molecule that
carries foreign DNA into a host cell,
replicates inside a bacterial (or yeast) cell
and produces many copies of itself and the
foreign DNA.
Features of cloning vectors
Types of Cloning Vectors
General steps of cloning any vector

23. Features of Cloning Vectors
1.Origin of replication: a sequence from
where replication starts.
2.Selectable marker: a method of selecting
for bacteria containing a vector with
foreign DNA; permits the growth of
transformants and eliminate the non-
transformants. usually accomplished by
genes encoding resistant to antibiotics
such as ampicillin,
chloramphenicol,tetracycline or
kanamycin.
3.Cloning site: to insert foreign DNA; the
most versatile vectors contain a site that

24. It shows a typical plasmid vector. It contains a polylinker which can recognize
several different restriction enzymes, an ampicillin-resistance gene (ampr) for
selective amplification, and a replication origin (ORI) for proliferation in the host
cell.

25. Plasmids or Bacteriophages are
used as vectors.
If we are able the link an alien DNA
with Bacteriophage or Plasmid
DNA, we can multiply its number
equal to the copy number of the
Plasmid or Bacteriophage.

26. Vectors
 DNA molecules capable of accepting a
‘foreign/new’ DNA fragment
 Most common are plasmids but could be of
other types also.
 They are self replicating because they have an
origin of replication (ori)
 Plasmids are circular, double-stranded, extra-
chromosomal pieces of DNA
 Plasmids carry antibiotic resistance genes and
a region to clone the ‘new’ DNA called the
multiple cloning site

27. PLASMID
 A plasmid is an independent, circular,
self-replicating DNA molecule that
carries only a few genes.

30. Vectors for cloning genes in
plants
•Agrobacterium tumefaciens can cause the plant disease
crown gall by transferring specific genes to the dicot
plant.
•A. tumefaciens contains a large plasmid called Ti
plasmid which can deliver T- DNA to transform normal
cell into a tumor and direct these tumor cells to produce
the desired chemicals.
•Plant genetic engineers have used this natural
transformation system as a vehicle for the introduction
of foreign DNA into plants.

31. Agrobacterium tumefaciens
is a soil bacteria that causes
common plant tumours, commonly
known as Crown Gall disease which
affects a wide variety of plants.
So what is Agrobacterium?????
A natural genetic engineer!!
The genome of Agrobacterium tumefaciens C58 has been
sequenced completely and consists of a circular chromosome,
a linear chromosome and two plasmids

32. A. tumefaciens gall is not a tiny thing!!!!!

33. Tumor-
producing
genes
Virulence region
Opine
catabolism
ORI
T-DNA
region
The Ti
Plasmid

34. During infection, the Ti plasmid is integrated
into the plant chromosomal DNA

38. Produce callus  transform callus 
stimulate shooting by cytokinin addition
Biology of Plants, Raven et. al., Freeman Worth Publishing, 1999
+ cytokinin
This procedure is easy
for dicotyledon plants
(legumes etc)

39. Monocotyledons are not easy to handle –
callus is very difficult to be initiated, and
A.tumefaciens is not pathogenic for them
1. Pericarp sholud be pulled back and
the immature embryos (0.5 - 1.0 mm) are
removed.
2. The immature embryos
are placed on
a callus induction medium
high osmotic media
prepare calli
for transfomation
Transformation
is performed
by gene gun method

40. After shooting calli are placed on a selective
media containing a herbicide for three weeks.
Then calli are transferred to a media
to induce the production of shoots.
After they form small shoots,
they are transferred to
DARKER containers on a root induction media.

41. Vectors for cloning genes in
Animals
 Retroviruses in animals have the ability to
transform the normal cells into cancerous
cells.
 Retroviruses are disarmed and now used to
deliver desirable genes into animal cells.

42. Competent Host
(for transformation with r-DNA)
 Chemical treatment: Bacteria cell is treated
by calcium to make them competent.
 Incubate the cells with r-DNA on ice,
 heat shock by placing them at 42oC,
 putting them back on ice.
This enables the bacteria to take up the r-DNA.

43. Other methods
 Micro injection: r-DNA is directly injected
into the nucleus of an animal cell.
 Gene gun or Biolistics: plant cells are
bombarded with high velocity micro-
particles of gold or tungstun coated with r-
DNA.
 Disarmed Pathogen: agrobacterium
tumifaciens in plants and Retroviruses in
animals.

44. Micro-injection: The host cell is immobilized by
applying a mild suction with a blunt pipette. The
foreign gene is then injected with a micro-injection
needle.

45. “Gene Gun” Technique
DNA coated
golden particles
Gene gun
Cell division
A plant cell with
the new gene
Transgenic plant
Plant cell
Cell’s DNA

46. DNA with desired gene and antibiotic resistance is
coated onto the surface of gold particles.
vacuum chamber
Calli are placed
in vacuum chamber,
Helium pressure
shot DNA into cells
Gene gun
Coating gold
particles with
DNA
Calli remain
on the high osmotic media
for 20 hours
following shooting.

47. Closer look on: “gene gun”

48. Processes of Recombinant
DNA Technology
 Isoaltion of the Genetic Material(DNA)
 Cutting of DNA at specific location
 Amplication of Gene of Interest using
PCR
 Insertion of r-DNA into the Host
cell/organism
 Obtaining the Foreign Gene Product

49. Isoaltion of the Genetic
Material(DNA)

51. Construction of a
Recombinant DNA
 Plasmid (autonomously replicating, circular,
extra-chromosomal DNA) is isolated.
 Plasmid DNA acts as a vector since it is
used to transfer the piece of DNA
attached to it to the host.
 Plasmid DNA also contains genes
responsible for providing antibiotic
resistance to the bacteria.

52.  Plasmid DNA was cut with a specific
restriction enzyme (‘molecular scissors’ −
that cut a DNA at specific locations).
 The DNA of interest (to be inserted)
was also cut with the same restriction
enzyme.
 The DNA of interest is hybridised with
the plasmid with the help of DNA ligase
to form a Recombinant DNA.

53.  Recombinant DNA is then transferred
to a host such as E.coli, where it
replicates by using the host’s replicating
machinery.
 When E.coli is cultured in a medium
containing antibiotic, only cells
containing recombinant DNA will be able
to survive due to antibiotic resistance
genes and one will be able to isolate the
recombinants.

55. Selecting cloned DNA molecules and
making more of them
Transformation and selection Plasmid multiplication

56. Polymerase Chain
Reaction (PCR)
 In this reaction, a small fragment of
deoxyribonucleic acid (DNA) or gene can be
rapidly cloned, or duplicated, to produce multiple
DNA copies. It requires:-
 Primers- small chemically synthesised
oligonucleotides that are complementary to the
regions of DNA.
 Taq Polymarase - Thermostable DNA
Polymerase isolated from Thermus aquaticus, a
heat-loving bacterium found in the hot springs of
Yellowstone National Park. It remain active during
the high temperature.
 Nucleotide Bases

57. Each cycle of PCR consists of three
phases
1. Denaturation: The DNA is heated to
cause its two linked strands to separate.
2. Annealing: The temperature of the
mixture is lowered to allow primers—
starter pieces of DNA—to bind to the
separated DNA.
3. Extension (Polymerization) : The Taq
polymerase enzyme extends the primer
using the nucleotides and copy the DNA
rapidly.

61.  One complete PCR cycle takes less than
two minutes to complete.
 Theoretically, the PCR cycle can be
repeated indefinitely, but the polymerase,
nucleotides, and primers are usually
renewed after about 30 cycles. .
 Thirty PCR cycles can produce 1 billion
DNA copies in less than three hours.

63. Applications of Recombinant DNA
Technology
1. To understand molecular events in biological processes such
as cell differentiation and aging.
2. It can be used to make precise gene maps.
3. Useful chemical compounds can be produced.
Example: injectable hepatitis B vaccine.
4. In the diagnosis of diseases.
Example:
a. Identification of food poisoning by Salmonella,
b. Hepatitis virus
c. HIV
5. Testing the DNA of parents who are carriers for genetic
disorders can be done and other chances of producing an
afflicted child can be predicted.