Maharashtra board XII th Science Chapter 12 Biotechnology Part 1

1. BIOTECHNOLOGY
By Dr. Janaki V. Pandey (PhD)
Chapter 12 (part 1)

2. Definition of Biotechnology
‘ The application of scientific and engineering
principles to the processing of materials by
biological agents to provide goods and service to
the human welfare’.

3. It uses scientific principles of
microbiology
 genetics
biochemistry
chemical engineering
Mathematics & statistics
computers
industrial processes.
Biological agent like plants and animal cells,
microorganisms, enzymes or their products.

4. Developmental phases of Biotechnology
Traditional biotechnology-
• It was based on fermentation technology using
microorganisms as in the preparation of curd,
ghee, vinegar, yogurt, cheese making, wine
making, etc.
Modern biotechnology-
• Recombinant DNA technology permits to modify
genetic material for getting new specific
products.
• The combination of biology & production
technology based on genetic engineering.

5. Features of technology that Differentiate
between modern & classical Biotechnology
1) Capability of science to change the genetic
material for getting new specific products through
rDNA technology, polymerase chain reaction (PCR),
microarrays, bioprocessing & cell culture and
fusions.
2) Ownership of technology and its socio- political
impact.
Various industries like pharmaceutical, food, agro, etc
are focussing attention to produce biotecholony- based
products.

6. Principles and Processes of
Biotechnology
● Genetic Engineering
Deals with alteration of genetic material (DNA &
RNA) & making a desired gene using invitro
process.
● Chemical Engineering
Deals with maintaining sterile environment for
manufacturing of useful products (Vaccine,
enzymes, antibodies, vitamins, etc.)

7. Recombinant DNA technology
It involves-
Repairing of defective gene by healthy genes.
Artificially synthesis of totally new gene.
Transfer of gene into new organism.
Manipulation of genes for improvement of
living organisms
 Combining of genes from two organisms
Altering the genotype
 Gene cloning .

8. Tools & Techniques for Gene Cloning
1. Instrumentation –
Gel permeation, osmotic
pressure, ion exchange
chromatography,
spectroscopy, mass
spectrometry,
electrophoresis, etc.
DNA, RNA, proteins, etc. vary in their molecular weight,
solubility, presence of charges, absorbance of light, etc.
Gel permetion chromatography

9. • It is the separation of charged molecules, applying an electric
field.
• It is applied for the separation of DNA, RNA and proteins.
• DNA being negatively charged, migrates to anode.
• Small fragments of DNA molecules, move faster and thus
separate faster.
• Use of Agarose gel electrophoresis, PAGE, SDA PAGE are the
different methods of electrophoresis.
a) Electrophoresis

10. Polymerase Chain Reaction (PCR) Machine

11. b) Polymerase chain reaction (PCR)
• PCR is used for gene cloning or gene
multiplication in vitro.
• PCR can generate a billion copies of the desired
segment of DNA or RNA, with high accuracy and
specificity, in a few hours.
• The process involves automatic thermal cycles for
denaturation & renaturation of ds DNA.
• The device required for PCR is called a thermal
cycler.

12. Mechanism of PCR
At the start of PCR,
the DNA segment
excess of two primer
molecules
four deoxyribonucleosides triphosphates
 the thermostable DNA polymerase are mixed
together in ‘eppendorf tube’ &
 the following steps are performed.

13. Steps of PCR
1. Denaturation-
The reaction mixture is heated to a temperature (90–
98o C)to separate two strands of desired DNA.
2. Annealing –
The mixture is allowed to cool (40–60o C) that permits
pairing of the primer to the complementary sequences
in DNA.
3. Primer Extension/ Polymerisation –
The temperature (70–75o C) allows thermostable Taq
(Thermus aquaticus) DNA polymerase to use single-
stranded DNA as template and adds nucleotides. It
takes around two minutes duration.

14. Steps of PCR
Replicating DNA
Strands using
Taq Polymerase
(70-75o C)

16. • One cycle takes 3 to 4 minutes.
• To begin second cycle, DNA is again heated to
convert dsDNA into ssDNA.
• The three steps are automatically repeated 20-30
times. Thus, at the end of ‘n’ cycles 2n copies of
DNA segments, are produced.
• Once the desired number of cycles is completed,
the amplified DNA segment is purified by gel
electrophoresis.
• After its sequencing, the amplified DNA segment
can be inserted into a cloning vector.
• Desired gene can also be obtained from gene
library.

17. 2. Biological tools
Enzymes
Cloning vectors (vehicle DNA)
Competent host (cloning organisms)

18. A. Enzymes
Different enzymes include
Lysozymes,
 Nucleases such as exonucleases,
endonucleases, restriction endonucleases,
DNA ligases,
DNA polymerases,
alkaline phosphatases,
reverse transcriptase, etc.

19. i) Restriction Enzymes
• Nuclease – cuts the Phosphodiester bonds.
• There are of 2 types
Exonuclease –cut nucleotides from the ends of
DNA strands.
 Endonuclease –cuts DNA from within.
• The restriction enzymes are molecular
scissors that are used to recognize and cut
DNA at specific sequences.

20. ii) Recognition sequences
• The sites recognized by restriction enzymes
are called recognition sequences or
recognition sites.
• They are 4 to 8 nucleotides long &
characterized by a particular type of internal
symmetry.
3’------- C T T A A G ------- 5’
5’------- G A A T T C ------- 3’
• When one reads the sequence in opposite
direction (3’ to 5’ or 5’ to 3’) it is
identical/same. It is called as palindrome.

21. • When the enzyme EcoRI attacks this
palindrome, it breaks each strand at the same
site in the sequence, which is indicated by the
arrow between the A and G residues.
3’ ---- C T T A A ↓ G ------5’
5’----- G ↑ A A T T C------ 3’
• Restriction enzymes either cut straight across
the DNA in the region of palindrome to give
blunt ends or cuts producing short, single
stranded projections at each end of DNA to
produce, cohesive or sticky ends.

23. B) CLONING VECTORS
DNA molecules which carry a foreign DNA
segment & replicate inside host cell are called
as cloning vector.
Eg: plasmids, bacteriophages, cosmid,
phagemids, BAC, YAC,
transposons, baculovirus
& MAC.

24. CHARACTERISTICS OF GOOD VECTOR
1. It should have the ability of independent replication.
2. It should be able to easily introduce into host cell.
3. It should have marker genes for antibiotic resistance.
4. It must contain unique cleavage site in one of the
marker gene for restriction enzyme.
5. It should have suitable control elements like
promoter, operator, ribosomal binding sites etc.

25. i) PLASMID
• The plasmids most commonly used in
recombinant DNA technology are those that
replicate in E. coli.
• Genetical modified plasmids are pBR 322,
pBR320, pACYA 177.

26. ii) PLASMID VECTORS FOR PLANTS
• Agrobacterium tumefaciens is a soil bacterium
• It causes a plant disease called crown gall, in the
plant characterized by a tumor.
• A. tumefaciens contains a Ti- plasmid having a
Gall producing gene T-DNA.
• The bacterium has ability to insert Ti plasmid in
the genome of infected plant.
• A. tumefaciens is most widely used for gene
transfer as it has a marker gene -Ti (tumor
inducing).
• Cry gene from Bacillus thuringenesis & Nif gene
from Rhizobium is cloned inside Agrobacterium
and then transferred to the other plants.

27. C) Competent host (cloning organisms)
• Organisms used for cloning are usually the
bacteria like Bacillus hemophilus, Helicobacter
pyroli and E. coli.
• Mostly E. coli is used for the transformation
with recombinant DNA
E. coli

28. METHODOLOGY FOR rDNA TECHNOLOGY
Steps involved are as follows:
1. Isolation of DNA from the
donor organism
2. Insertion of desired foreign
gene into cloning vector.
3. Transfer of rDNA into
suitable competent host
4. Selection of the transformed
host cell
5. Multiplication of the host
cell.
6. Expression of the gene to
obtain the desired product

29. a) Isolation of DNA from the donor organism
• The desire gene of the donor organism are sheared
with the blender & treated with suitable detergent.
Genetic material from the donor is removed isolated
and purified by using several techniques. Isolated
DNA can be spooled on to a glass rod.
• Isolated DNA is cleaved by Restriction
Endonucleases (RE) at specific sites.
• This breaks the DNA into fragments. Cleaved DNA
fragments have cohesive, sticky, staggered ends or
blunt ends.
• From cleaved DNA fragments desired gene is isolated
and selected for cloning.
• This is now called foreign DNA or passenger DNA.

30. b) Insertion of desired foreign gene into
cloning vector.
• Plasmids are isolated from the vector organisms.
It is cleaved by same RE which was used for
desired gene.
• The foreign/desired DNA is inserted into a cloning
vector eg. pBR 322.
• Foreign DNA is joined into the vector DNA by
using enzyme DNA ligase.
• The combination of vector DNA and foreign DNA
is now called Recombinant DNA or Chimeric DNA
& the technology is referred to as rDNA
technology.

31. c) Transfer of rDNA into suitable competent host
• The rDNA is transferred into a competent host
i.e Bacterium.
• Host cell takes up the naked rDNA by the
process of transformation & incorporates into
it own chromosomal DNA.
• Now the expression of the trait are controlled
by the foreign DNA.
• The competent host is called as transformed
cell.
• The bacteria used are E.coli & A. tumefaciens.

32. • Foreign DNA can also be transferred directly
into the naked cell or protoplast of the
competent host cell, without using vector.
• This is done by using techniques like
electroporation, microinjection, lipofection,
shot gun, ultrasonification, biolistic method,
etc.
• But in plant biotechnology the transformation
is through Ti plasmids of A. tumefaciens.

33. d) Selection of the transformed host cell
• The transformation process generates a mixed
population of transformed (recombinant) and
non-transformed (non-recombinant) host cells.
• For isolation of recombinant cell from non-
recombinant cell, marker gene of plasmid vector
is employed. For example, pBR 322 plasmid
vector contains different marker gene (Ampicillin
resistant gene and Tetracycline resistant gene).
• When pst1 RE is used, it knocks out Ampicillin
resistant gene from the plasmid, so that the
recombinant cell become sensitive to Ampicillin.

34. e) Multiplication of transformed host cell
• The transformed host cell are introduced into
fresh culture media.
• At this stage the host cell divides & redivide
along with the rDNA carried by them.

35. f) Expression of the gene to obtain the
desired product
• The rDNA multiplies in the host and is
expressed as a protein. This is now a
recombinant protein.
• The Protein is separated and purified through
downstream processing using suitable
bioreactor.
• The desired product like alcohol, enzymes,
antibiotics, etc. are produced.

36. Application of Biotechnology
BIOTECHNOLOGY
ANIMAL BIOTECHNOLOGY
Tissue culture, generative
medicine, immnotechnology,
GM animals,
pharamacogenesis,
gene therapy
INDUSTRIAL BIOTECHNOLOGY
Enzyme biotech, protein &
metabolic engineering,
metabolomics
PLANT BIOTECH
Cell & tissue
culture, GM crops,
marker assisted
selection
MICROBIAL BIOTECH
Microbial genomics,
biofertilizers,
biopesticides, IPM
GENOMICS
Whole genome
sequencing, gene
function, reverse
genetics, bioinformatics
ENVIRONMENTAL BIOTECH
Pollution control, biofuels,
bioremediation, biodiversity

37. a) HEALTH CARE BIOTECHNOLOGY
Sr.
No
HEALTH CONDITIONS RECOMBINANT PROTEIN
1 Anaemia Erythropoetin
2 Asthma Interleukin-1 receptor
3 Atherosclerosis Platelet derived growth factor
4 Parturition Relaxin
5 Blood clots Tissue plasminogen Activator (TPA),
Urokinase
6 Cancer Interferons, tumor necrosis factor
interleukins,
Macrophage activating factor
7 Diabetes Insulin
8 Emphysema α1- Antitrypsin
9 Haemophilia A Factor VIII
10 Haemophilia B Factor IX
11 Hepatitis B Hepatitis B vaccine

38. Human Insulin
• Insulin is a hormone produced by 𝛽- cells of islets of
Langerhans of pancreas.
• It control blood sugar levels in humans.
• Diabetes mellitus is a disease in which some people
cannot make insulin themselves.
• DNA sequence of insulin for two chains A and B are
chemically synthesized & separately inserted into two
pBR322 plasmid vector.
• The genes are inserted by the side of β-galactosidase
gene of the plasmid.
• The recombinant plasmids were then separately
transformed into E. coli host.
• The host produced penicillinase + pre-pro insulin.
Insulin is later separated by trypsin treatment.