CHAPTER : 9 BIOTECHNOLOGY : PRINCIPLES & PROCESSES

Page No. : 162-175

Total Pages : 14
Questions Asked : 55

2-4 QUESTION PER PAGE AT AN AVERAGE

BUT, LETS SEE WHICH PAGE HAS MAXIMUM WEIGHTAGE

• Which page has the maximum

165, 168 = 2 Page
37.2% QUESTIONS

2010-2024 Page No.

0 163

10,11,12,16,17,18,
2 164,166,172,175
20,21(3),22

12,14,20,16,10,19 3 173, 174

12(2),19,20,21
4 169
22(2)
11(2),12,13,14,16,17,
19(2),20,21,22(2),24 5 167, 170, 171

10(2),12,13,15(2)
8 165, 168
16(2), 20,21,22(2)
BIOTECHNOLOGY : PRINCIPLES AND PROCESSES

1. Biotechnology deals with techniques of using live organism or

enzymes from organisms to produce products & processes
useful to humans.
2. Today genetically modified organism to achieve the same on
large scales.
3. In-vitro fertilisation leading to a test tube baby, synthesising a
gene & using it, developing DNA vaccine or correcting a
defective gene, all are part of biotechnology.
4. The European Federation of Biotechnology (EFB) given the
definition of Biotechnology & included both traditional view &
modern molecular biotechnology

DEFINED AS FOLLOWS :

“The integration of natural science & organisms, cell, parts there

of & molecular analogues for products & services.
5. Two core techniques:
1. Genetic engineering: Alter the chemistry of genetic material
ð Introduce into the host organisms ð Change the
phenotype of the host organism.
. 2. Bioprocess engineering: Maintenance of sterile
(microbial contamination free) ambience in chemical
engineering process to enable growth of only desirable
microbes.
6. Traditional hybridisation: Inclusion & multiplication of
undesirable genes along with the desired genes.
7. Genetic engineering: Create recombinant DNA, used of
gene cloning & gene transfer.
(NEET-2012)[NCERT 164]
• Allow us to isolate & introduce only one or a set of desirable
genes without introducing undesirable genes into the target
8. The construction of the first recombinant DNA emerged from
the possibility of linking a gene encoding antibiotic resistance
with native plasmid of salmonella typhimurium by Stanley
cohen & Herbert Boyer in 1972.
(NEET-2020)[NCERT 164]
9. Plasmid : Autonomously replicating circular extra-chromosomal
DNA.
(NEET-2016)[NCERT 164]
10. Cutting of DNA at specific location become possible with
Restriction endonucleus (molecular scissors) & linking of cut
DNA possible by enzyme DNA ligase.
11. The ability to multiply copies of antibiotic resistance gene in
[Link] was called cloning.
(NEET-2018, 2015)[NCERT 165]
12. Three basic steps in genetically modified on organism:
1. Identification of DNA with desirable genes.
2. Introduction of identified DNA within the host
3. Maintenance of introduced DNA into the host & transfer of the
DNA to its progeny. (NEET-2024)

TOOLS OF RECOMBINANT DNA TECHNOLOGY

• Enzyme (Restriction enzyme, polymerase enzyme, ligase)
• Vectors
• Host organism
13. In 1963, two enzyme responsible for restricting the growth of
Bacteriophage in Escherichia coli.
1. Added methyl group to DNA
2. Cut DNA
14. First restriction endonuclease discovered was Hind-II.
15. Hind II always cut DNA molecules at a particular point by
recognising a specific sequence of six base pairs called
recognition sequence.
(NEET-2016, 2024)[NCERT 165]
16. Today we know more than 900 restriction enzyme, isolated
from over 230 strains of bacteria each with different
recognition sequences.
17. Convention for naming these enzymes. First letter comes from
the genus & second two letter comes from the species of
prokaryotic cell. The fourth letter indicates strain of bacteria.
Roman number indicates the order in which enzyme were
isolated from that strain of Bacteria.
18. Restriction enzyme belong to a larger class of enzyme -
Nucleases
Nucleases : Exonucleases (Remove nucleotides from the
ends of DNA) & Endonucleases (cut at specific
positions within the DNA).
(NEET-2020, 2024)[NCERT 166]
19. The restriction site of these enzyme is a specific palindromic
nucleotide sequence in the DNA.
(NEET-2022, 2019)[NCERT 166]
20. Restriction enzyme cut the strand of DNA either little away from
the centre of the palindrome site or in the centre of palindrome
site & produce either sticky ends or Blunt ends of DNA.
[RE-NEET-2024] (NEET-2019)[NCERT 167]
21. Palindromic DNA sequence : Read some on the two strands
when orientation of reading is kept the same.
eg: 5’ - GAATTC - 3’
[RE-NEET-2024] (NEET-2022, 2021, 2020,2019,2012)
3’ - CTTAAG - 5’
[NCERT 167]
22. Stickiness of the ends facilitates the action of the enzyme DNA
ligase.
(NEET-2017, 2020)[NCERT 167]
23. Restriction endonuclease are used in genetic engineering to
form “recombinant molecule of DNA, composed of DNA from
different sources/genomes.

Vector
DNA
Foreign DNA (plasmid)

Same restriction enzyme cutting both foreign

DNA and vector DNA at specic point

Ligases join foreign

DNA to plasmid

Recombinant DNA
Molecule

Transformation

[Link]
Cells divide
(Cloning Host)

Diagrammatic representation of recombinant DNA technology

24. Unless one cuts the vector & the source DNA with the some
restriction enzymes the recombinant vector molecule can not be
created.
SEPERATION & ISOLATION OF DNA FRAGMENTS
[NCERT 168]
25. DNA fragments can be seperated by Gel electrophoresis.
(NEET-2013)
26. DNA fragments negatively charged & move toward the anode
under an electric field through a medium/matrix.
(NEET-2019, 2017)[NCERT 168]
27. Most commonly used matrix is Agarose (Natural polymer
extracted from sea weeds). [RE-NEET-2024]
28. DNA fragments seperate (resolve) according to their size
through seiving effect provided by the agarose gel.
[RE-NEET-2024]
29. Smaller the fragment size, the further its moves.
(NEET2019)[NCERT 168]
30. Separated DNA fragments Ethidium bromide followed by
exposed to UV radiation.
(NEET-2022, 2020, 2019, 2017)[NCERT 168]
31. Bright orange coloured bonds of DNA is a ethidium bromide
stained get exposed to UV light.
(NEET-2022, 2021, 2017,23)[NCERT 168]
32. The separated band of DNA are cut out from the agarose gel &
extracted from the get piece - Elution.
[NEET-2022][NCERT 168]
33. Coloning vectors : Plasmids Bacteriophage
34. Bacteriophage because of their high number per cell, have very
high copy number of their genome within the bacterial cells.
35. Some plasmids may have only one or two copies per cell
whereas other may have 15-100 copies per cells.
36. A vector must have following three featured :
1. Origin of Replication (Ori) : Start replication & control
copy number of linked DNA
[NEET-2022][NCERT 169]
2. Selectable marker : Identifying & eliminating non
(NEET-2019) transformant & selectively
permitting the growth of
[NCERT 169] transform-ants.
37. Transformation : Procedure through which a piece of DNA is
introduced in a host bacterium.
38. Genes encoding resistance to antibiotics that useful selectable
marker for [Link] Ampicillin, Chloram-phenicol, Tetracycline,
Kanamycin.
(NEET-2022, 2012)[NCERT 169]
3. Cloning Sites : Vector needs to have very few, preferably
single recognition sites for commonly used restriction
enzymes.
[NEET-2022][NCERT 169]
39. Presence of more than one recognition sites within the vector will
generate several fragments, which will complicates the gene
cloning.
Hind III [NEET-2022][NCERT 169]
EcoR I Cla I
Pvu I
Pst I BamH I
ampR R
tet
pBR322 Sal I

ori
rop

Pvu II (NEET-2021, 2012)[NCERT 169]

40. Rop codes for the proteins involved in the replication of the
plasmid.
41. Differentiation of recombinants from non-recombinants on the
basis of their ability to produce colour in the presence of
chromogenic substrate.
42. Presence of chromogenic substrate gives blue coloured
colonies if the plasmid in the bacteria do not have an insert.
[NEET-2022][NCERT 170]
43. Presence of insert results into insertional inactivation of the B
galactosidase gene & the colonies do not produce any colour,
these are identified as recombinant colonies.
(NEET-2013)[NCERT 170]
44. Retroviruses in animals have the ability to transform normal cell
into cancerous cells. (NEET-2018)[NCERT 170]
45. Ti Plasmid (Tumor Inducing) Agrobacterium tumifaciens is
modified into cloning vector. (NEET-2024)[NCERT 170]
46. DNA is the hydrophilic molecule, it can not be pass through
cell membrane.
47. In order to force bacteria to take up plasmid, the bacterial cell
must first be made “competent” to take up DNA.
48. For making competent, treating them with a specific
concentration of a divalent cation, such as calcium (Increase
efficiency with which DNA enters the bacterium through pores in
its cell wall).
0
49. Recombinant DNA placed 1st on ice then at 42 C (Heat shock)
and then putting them back on ice. This enables the bacteria to
take up recombinant DNA.
50. Introduction of alien DNA into Host cell via:
Micro injection (For Animals) : Recombinant DNA directly
injected into the nucleus of an animal cell.
Biolistics or Genegun (for plants): Cells are bombarded with
high velocity micro-particles of Gold or Tungsten coated
with DNA (2012,23)[NCERT 171]
 Disarmed pathogen : Allowed to infect the cell, transfer the
recombinant DNA into the host.
PROCESSES OF RECOMBINANT DNA TECHNOLOGY
Steps:
[NEET-23][NCERT 171]
1. Isolation of DNA
2. Fragmentation of DNA by restriction endonuclease
3. Isolation of desired DNA fragment
4. Ligation of DNA fragment into a vector
5. Transferring the recombinant DNA into the host
6. Culturing the ;host cells in a medium at large scale &
extraction of the desired product.
51. Bacterial cell wall isolated with enzyme- Lysozyme.
52. Plants cell isolated with enzyme cellulase
53. Fungus cell isolated with enzyme chitinase
54. Purified DNA ultimately precipitates out after the addition of
chilled ethanol. This can be seen as collection of fine thread in
the suspension- Spooling.
(NEET- 2021, 2019, 2013,23)[NCERT 171]
PCR (Polymerase Chain Reaction): Amplification of gene of
Interest
Steps: Denaturationð Annealingð Extension.
[RE-NEET-2024] (NEET-2022, 2021, 2018)[NCERT 172]
55. Multiple copies of gene (or DNA) of interest is synthesised in
vitro using two set of primers (Small chemically synthesises
oligonucleotides that are complementary to the regions of
DNA) & enzyme DNA polymerase.
56. If the process of replication of DNA is repeated many times, the
segment of DNA can be amplified to approx. Billion times (1
Billion copies).
57. Thermostable DNA polymerase, which remains active during the
high temperature induced denaturation of ds DNA.
[RE-NEET-2024] (NEET-2021)[NCERT 173]
58. Thermus aquaticus (Bacteria) active at high temperature.
(NEET-2020)[NCERT 173]
59. If any protein encoding gene is expressed in a heterologous
host, it is called Recombinant Protein.
• In continuous culture system, used medium is drain out from
one side & fresh medium is added from other side to maintain
the cell in their Physiologically most active
log/exponential phase.
• To produce in large quantities, the development of
Bioreactors are required (NEET-2019)[NCERT 174]
• Large volumes (100 -1000 liters) of culture can be processed
was required.
• Bioreactors : A vessels in which raw material are biologically
converted into specific products, individual enzymes etc,
using microbial plants, animal or Human cells.
60. A bioreactor provides the optimal conditions for achieving the
desired product by providing optimum growth conditions :-
Temperature, pH, Substrate, Salts, Vitamines, Oxygen.
61. The most commonly used Bioreactors are of stirring type.
(NEET-2024)
62. A stirred tank reactor is usually cylindrical or with a curved
base to facilitate the mixing of the reactor contents
(NEET-2016)[NCERT 174]
63. Stirrer facilitates even mixing & oxygen availability throughout
the bioreactars.
64. Bioreactoar has : Agitator system, oxygen delivery system,
foam control system temperature control system, pH control
system, sampling ports. (NEET-2024)
65. After completion of Biosynthetic stage, the product has to be
subjected through a series of processes before it is ready for
marketing process are :–
(NEET-2016, 2017)[NCERT 175]
Seperation, purification, Preservation, Clinical trials
66. Down stream processing & quality control testing vary from
product to product.