This presentation is all about biotechnology. It is about the basic aspects of Biotechnology and covers a lot of topics under biotechnology, recombinant DNA technology. This is specifically for the HSC students of Mumbai. I hope that it helps.

2. Definition
• Biotechnology is the
integration of natural
science and organisms,
cells, parts thereof and
molecular analogues for
products and services.

3. Oldest form of biotechnology

4. Application of
fermentation in
production of wine and
other alcoholic
beverages is also a
biotechnological
technique

5. But with time biotechnology
gradually became more
sophisticated.

6. DNA
manipulation
Protein Tissue
engineering culture
Biotechnology
Immobilized Protoplast
enzymes fusion
Cell
catalysis

7. Biotechnology led to production of many products and
provides many services for human welfare.

9. Dragon Fly The biotechnology
industry has
mushroomed since
1992, with U.S.
health care biotech
revenues from
publicly traded
companies rising
from $8 billion in
1992 to $58.8
billion in 2006.
There were 180,000 people employed by U.S. biotechnology companies in
2006.
There are more than 400 biotech drug products and vaccines currently in clinical trials targeting
more than 200 diseases, including various cancers, Alzheimer’s disease, heart disease, diabetes,
multiple sclerosis, AIDS and arthritis.

11. In-Vitro Fertilization
Also called as
Test tube baby

12. Plant tissue culture
DNA vaccines

13. The recombinant DNA technique was
first proposed by
Peter Lobann A. Dale Kaiser

14. The present day rDNA technology flourished after
the work of

15. Gene coding for
Plasmid antibiotic resistance
Vector
Cloning
Salmonella typhimurium
They successfully linked a gene
coding for antibiotic resistance with
a native plasmid of Salmonella
typhimurium with the vector plasmid
and then cloning it in E.coli.
E. coli

16. What is recombinant DNA ?
• Technique of manipulating the genome of a cell
or organism so as to change the phenotype
desirably.
Seedless guava Calorie free sugar

17. Introducing
in Host
Isolating Culturing the
genomic cells
DNA
Insertion of
DNA in a
vector
Transformation
Fragmenting of host cell
this DNA Screening
the
fragments

18. Isolating Isolating
genomic genomic DNA
DNA
from the donor.
Fragmenting
Fragmenting this DNA using
this DNA molecular
scissors.

19. Screening the
Screening
the fragments for a
fragments “desired gene”.
Inserting the
Insertion of
fragments with the
DNA in a desired gene in a
vector „cloning vector‟.

20. Introducing the recombinant
Introducing vector into a competent host
in Host
cell
Culturing these cells to obtain
Culturing multiple copies or clones of
the cells desired DNA fragments
Using these copies to
transform suitable host cells
Transformation so as to express the desired
of host cell gene.

21. Example: Production of Insulin

22. Tools used in recombinant
DNA technology
• Enzymes
• Vectors

23. Tools used in recombinant
DNA technology
• Enzymes
 Act as biological scissors.
 Most commonly used are:
 Restriction endonuclease
 DNA ligase
 DNA polymerase
 Alkaline phosphatases

24. Tools used in recombinant
DNA technology
• Vectors
 Low molecular weight DNA molecules.
 Transfer genetic material into another
cell.
 Capable of multiplying independently.

25. Vector
Bacteriophage
DNA
Artificial
Plasmid Vector DNA
Cosmid

26. Insertion of vector in target
cell is called
• Bacterial cells – Transformation
• Eukaryotic cells – Transfection
• Viruses - Transduction

27. Insertion of vector in target
cell
Vectors used:
• Bacteria- plasmids, cosmid,
lambda phage
• Insects- baculoviruses
• Plants- Ti plasmid
• Yeast cells- YAC (yeast artificial
chromosome)

28. HOST DONOR
DNA DNA
Fragmented by Restriction Endonuclease
DNA strands with sticky ends
Sticky ends base pair with complementary sticky ends
DNA ligase links them to form rDNA
Cloned
In vitro In vivo
Polymerase chain Prokaryotic or eukaryotic cell,
reaction (PCR) mammalian tissue culture cell

29. Some examples of therapeutic products
made by recombinant DNA techniques
¶ Blood Proteins: Erythropoietin, Factors VII, VIII, IX; Tissue
plasminogen activator; Urokinase.
¶ Human Hormones: Epidermal growth factor; Follicle
stimulating hormone, Insulin.
¶ Immune Modulators: α Interferon, β Interferon; Colony
stimulating hormone; Lysozyme; Tumor Necrosis factor.
¶ Vaccines: Cytomegalovirus; Hepatitis B; Measles; Rabies

30. Transposons
• Transposons are sequences of
DNA that can move or transpose
themselves to new positions
within the genome of a single
cell.
• Also called „Jumping genes‟.

31. • 1st transposons were discovered
by
Barbara
McClintock
in Zea mays (maize)

32. Types of transposons
• According to their mechanism
they are classified as:

33. Retrotransposons
• Follows method of “Copy and
Paste”.
• Copy in two stages.
DNA RNA DNA
Transcription Reverse
Transcription

34. DNA transposons
• Follows the method of “Cut and
Paste”.
• Do not involve RNA intermediate.
Enzyme Transposase
Cuts out transposon
Ligates in new position

35. Plasmid
• Plasmids are small, extra chromosomal, double
stranded, circular forms of DNA that replicate
autonomously.
• The term was introduced by in 1952.
Joshua Lederberg

36. Plasmid
• Found in bacterial, yeast and occasionally in
plants and animal cells.
• Transferable genetic elements or
‘Replicons’.
• Size- 1 to 1000 kilo bp.
• Related to metabolic activity.
• Allows bacteria to reproduce under
unfavorable conditions.

37. Plasmid
Nomenclature
Lower case P (p)
First letters of researchers name or place where
it was discovered.
Numerical numbers given by workers.

38. Plasmid
Eg. Plasmid pBR 322
BR is for Bolivar and Rodriguez, who designated it as 322

39. Plasmid
Eg. Plasmid pUC 19
UC stands for University of California

40. Plasmid- Cosmids
• Cosmids are plasmids with cos sequence.
• They are able to accommodate long DNA fragments
that plasmids can’t.

41.  A bacteriophage is a virus that infects bacteria.
 Virulent portion is deleted.
Genetic material can be
ssRNA, dsRNA, ssDNA,
dsDNA.

42.  For Single genes- Plasmids are used
 For Large pieces of DNA- Bacteriophages

43.  48.5 kb in length.
 Cos sites of 12 bp at the ends.
 Cohesive ends allow circularizing DNA in host.

44. (1) Phage attaches to a specific host
bacterium.
(2) Injects its DNA,
(3) Disrupting the bacterial genome
and killing the bacterium, and
(4) Taking over the bacterial DNA and
protein synthesis machinery to
make phage parts.
(5) The process culminates with the
assembly of new phage, and
(6) The lysis of the bacterial cell wall
to release a hundred new copies of
the input phage into the
environment.

45. RESTRICTION FRAGMENTS
A restriction fragment is a DNA fragment resulting
from the cutting of a DNA strand by the restriction
enzyme.
 Process is called restriction.

46. RESTRICTION FRAGMENTS
 Steward Linn along with Werner Arber in 1963
isolated two enzymes.
 One of them is Restriction Endonuclease.
 Restriction Endonuclease can cut DNA.
 Restriction Endonuclease are basic requirement
for gene cloning or rDNA technology.

47. RESTRICTION FRAGMENTS
Nucleases
They remove They make cuts
nucleotides at specific
Exonuclease Endonuclease positions within
from the ends
of the DNA the DNA

48. TYPES OF REN
REN
Type I Type II Type III
Mostly used in rDNA technology.
More than 350 types of type II
endonucleases with recognition sites are
known.
Can be used to identify and cleave within

49. NOMENCLATURE OF REN
 First letter- genus name of bacteria (in italics).
 Next- first two letters of the species name (in
italics).
 Next- strain of the organism.
 Roman number- order of discovery.

50. NOMENCLATURE OF REN
 Eg. - EcoR I
E- Escherichia, co- coli, R-strain Ry 13,
I- first endonuclease to be discovered.
 Eg.- Hind III
H- Haemophilus, in- influenzae, d- strain Rd,
III- third endonuclease to be discovered.

51. RECOGNITION SEQUENCE (RESTRICTION SITES)
 It is the site/ sequence where REN cuts the DNA.
 Sequence of 4-8 nucleotides.
 Most restriction sites are Palindromes.

52.  In DNA, palindrome is a sequence of base pairs
that reads the same on the two strands when
orientation of reading is kept same.

53. CLEAVAGE PATTERNS OF REN
 REN recognizes the
restriction site.
 Cleave the DNA by
hydrolyzing
Phosphodiester bonds.
 Isolate a particular gene.
 Single stranded ends
called sticky ends.

54.  These sticky ends can
form hydrogen bonded
base pairs with
complementary sticky
ends or any other cleaved
DNA.

55. CLEAVAGE PATTERNS OF REN

56. Gel
 Restriction fragments electrophoresis
yield a band pattern
characteristic of the
original DNA molecule &
restriction enzyme used.
Bands

57. PREPARING AND CLONING A DNA LIBRARY
 Collection of DNA fragments from a particular species that
is stored and propagated in a population of micro
organisms through molecular cloning.

58. GENOMIC LIBRARY
 Collection of all clones of DNA fragments of complete
genome of an organism.
 All DNA fragments are cloned and stored as location of
desired gene is not known.
 Screening of DNA fragments can be done by
Complementation Or by using Probes.

59. Construction of Genomic Library.
Entire genome isolated
Cut into fragments by REN
Fragments inserted in Vector
Recombinant vectors are transferred into
suitable organism
Transferred organisms are cultured and
stored

60. CDNA LIBRARY
 cDNA is Complementary DNA.
 Produced using Teminism i.e. Reverse Transcriptase.
 Constructed for eukaryotes.

61. cDNA is made from mRNA
AAAAAAA Mature mRNA
Start Stop
TTTTTTT
Add polyT primer, nucleotides, and
Reverse Transcriptase
AAAAAAA DNA/RNA
TTTTTTT
RNA removed (by NaOH) and
second strand synthesized
TTTTTTT
Complementary DNA cDNA

62. Gene Amplification (PCR)
 It is obtaining multiple copies of a known DNA
sequences that contain a gene.
 Done artificially by using PCR (Polymerase Chain
Reaction)

63. PCR (Polymerase Chain Reaction)
 Developed by in 1983.
Kary Mullis
 In Vitro technique.
 Scientific technique to generate billions of copies of a
particular DNA sequence in a short time.

64. PCR Machine

65. Requirements for PCR technique
Primers-forward and
A DNA segment
DNA segment Primers reverse, are
100-35,000 bp in synthetic
length to be oligonucleotides and
amplified. complementary to
PCR the desired DNA
segment
Four types of Thermostable
deoxyribonucleotid dNTPs DNA Enzyme that can
es i.e. dCTP, dGTP, polymerase withstand upto 94°
dTTP, dATP C.

66. Steps of PCR technique
The double strand melts open to single
stranded DNA, all enzymatic reactions stop
(for example : the extension from a previous
cycle).
Ionic bonds are constantly formed and
broken between the single stranded primer
and the single stranded template. Once
there are a few bases built in, the ionic bond
is so strong between the template and the
primer, that it does not break anymore.
The bases (complementary to the template)
are coupled to the primer on the 3' side (the
polymerase adds dNTP's from 5' to 3',
reading the template from 3' to 5' side,
bases are added complementary to the
template)

67. The exponential amplification of the gene in
PCR.

68. Application of biotechnology in
agriculture- Bt crops

69. Bacillus thuringiensis
• Soil bacterium.
• Produces a protein that has
insecticidal properties.
• Traditionally used as spray.

70. Mechanism of Bt Bt (in inactive form)
sprayed on Crops
• Bt produces Bt toxins which
are inactive protoxins. Eaten by insect
• When an insect ingests it,
inactive protoxin gets Toxin gets activated by
alkaline pH of insect’s gut
converted into active form due
to alkaline pH of the insect’s
gut. Swelling of gut of insect
• This led to swelling of gut and
ultimately death of insect
Death of insect

71. Crop plants are now
engineered to express
Bt toxin.
• Cry gene in Bt produces
inactive protoxins.

72. Bt crops are now commercially available.
For Eg.
Bt Corn Bt Rice Bt Cotton Bt Tomato
Bt Brinjal Bt Soybean Bt Potato

73. Agrobacterium tumefaciens
• Soil bacterium.
• Causes crown gall tumors in
dicotyledonous plants.
• T DNA (gall producing gene) occurs
in Ti plasmid.
• Ti plasmid is used as vector for Mechanism
higher plants.
• Many genetically modified plants are
produced using A. tumifaciens.
Tumor

74. Ti Plasmid

75. • Desirable genes such as Cry gene an Nif gene is
cloned inside A. tumifaciens and then transferred into
another plant.
Nif Gene
isolated from
Rhizobium

76. Examples
1. Flavr savr tomato 2. Golden Rice
 Longer shelf life.  Greater pro vitamin A content.
 Antisense DNA is introduced that  Genetically engineered.
retards ripening

77. Bio-Safety Issues
Biosafety
issues
Impact on
human health Genetically Impact on
and modified Agriculture
environment
organisms
Ethical
issues

78. Genetic modification of organisms
can lead to
 Contamination of gene pools.
 Consumption may lead to allergies.
 Hazardous microbes may escape
laboratory
Therefore manipulation of organisms
needed regulation

79. Genetic Engineering Approval
Committee
In India, GEAC takes decision regarding validity of GM
research and introduction of GM products.

80. Biopiracy
 The patenting of plants, genes, and other biological
products that are indigenous to another country
 Developed countries patent the knowledge and
resources of underdeveloped countries and enjoy
immense profits.

81. Biopatent
A patent is granted
by the government
to the inventor for
biological entities,
processes and
products.

82. Case Study
Texmati was derived
by crossing Indian
Basmati rice with a
semi dwarf variety.
A Texas based
company got patent
on rights of basmati.
Indian Basmati rice
Texmati rice

83. Some other Examples
Turmeric Neem Margosa

84. What can be done?
Genetic Literacy
Movement in Schools
and Colleges on rapid
developments in
Molecular Genetics

85. What will it do?
Better understanding of opportunities and
risks of rDNA technology.
Promote safe and responsible use of tools
of genetic engineering.