1) Gene expression in eukaryotes is regulated through various mechanisms including chromatin remodeling, enhancers and repressors, locus control regions, gene amplification, gene rearrangement, alternative RNA processing, class switching, and mRNA stability. 2) Key differences in prokaryotic and eukaryotic gene expression include larger eukaryotic genomes, different cell types requiring gene regulation, lack of operons, chromatin structure, and uncoupling of transcription and translation. 3) The lac operon in E. coli is regulated through negative control in the absence of lactose, double negative control in the presence of only lactose, and absence of positive control in the presence of both glucose and lactose.

1. Molecular Biology
Revision series-3
Namrata Chhabra
MHPE, MD, MBBS, FAIMER FELLOW
Gene expression and Recombinant DNA
Technology

3. Gene Expression
o Gene expression is the
combined process of the
transcription of a gene into
mRNA,
o the processing of that
mRNA, and
o its translation into protein
(for protein-encoding genes).
17-Jan-21 3

4. Significance of Gene Expression
Regulated expression of
genes is required for
❑ Adaptation,
❑ Differentiation and
❑ Development
17-Jan-21 4

5. Types of gene regulation
There are three types of genes
regulation-
• Positive
• Negative and
• Double negative
17-Jan-21 5

6. Types of gene regulation
A) Positive regulation
• When the expression of genetic information is quantitatively
increased by the presence of a specific regulatory element, regulation
is said to be positive.
• The element or molecule mediating positive regulation is a positive
regulator or activator.
17-Jan-21 6

7. Types of gene regulation
B) Negative regulation
• When the expression of genetic information is diminished by the
presence of a specific regulatory element, regulation is said to be
negative.
• The element or molecule mediating negative regulation is said to be a
negative regulator or repressor.
17-Jan-21 7

8. Types of gene regulation
A double negative has the effect of acting as a positive.
• An effector that inhibits the function of a negative regulator will bring
about a positive regulation.
• Many regulated systems that appear to be induced are in fact
derepressed at the molecular level.
17-Jan-21 8

9. • In prokaryotes, the genes
involved in a metabolic
pathway are often present in a
linear array called an Operon,
e.g., the lac Operon.
• An Operon can be regulated
by a single promoter or
regulatory region.
• The cistron is the smallest
unit of genetic expression.
17-Jan-21 9
Features of Prokaryotic gene Expression

10. Lac Operon Model
• Jacob and Monod in 1961 described their Operon model in a classic
paper.
• Their hypothesis was to a large extent based on observations on the
regulation of lactose metabolism by the intestinal bacterium E coli.
17-Jan-21 10

11. Lac Operon – Basic concept
• Bacteria such as E. coli usually rely on glucose as their source of carbon
and energy.
• However, when glucose is scarce, E. coli can use lactose as their carbon
source even though this disaccharide does not lie on any major
metabolic pathways.
• An essential enzyme in the metabolism of lactose is β-galactosidase,
which hydrolyzes lactose into galactose and glucose
17-Jan-21 11

12. Lac Operon – Basic concept
Action of Beta galactosidase on lactose, breaks lactose to galactose
and glucose
17-Jan-21 12

13. 17-Jan-21 13
Components of Lac Operon

14. Lac Operon Gene Gene function
Lac I Constitutive gene synthesizes lac repressor constantly
Lac Z Gene for -galactosidase subunit
Lac Y Gene for Permease subunit
Lac A Gene for Thiogalactoside transacetylase subunit
Promoter or P Site for RNA polymerase binding & initiator of transcription
Operator or O Repressor binding site
Components of Lac Operon
17-Jan-21 14

15. How does the lac repressor inhibit the
expression of the lac Operon?
•The lac repressor can exist as a
dimer of 37-kd subunits, and two
dimers often come together to form
a tetramer.
• In the absence of lactose, the
repressor binds very tightly and
rapidly to the operator.
17-Jan-21 15

16. Negative Control- Repression
17-Jan-21 16

17. Lac I
Promoter
gene
Operator
gene
Lac Z Lac Y Lac A
R
Translation &
Transcription
RNA polymerase
No Gene
Expression
17-Jan-21 17

18. (b) Double negative control- Derepression
17-Jan-21 18

19. Lac I
Promoter
gene
Operator
gene
Lac Z Lac Y Lac A
RNA polymerase
Repressor
tetramer
R
Translation &
Transcription
Inactive
repressor
Desired
product
17-Jan-21 19

20. c) Positive control- CAP-cAMP binding
❑There are also DNA-binding proteins that stimulate transcription.
❑One particularly example is the catabolite activator protein (CAP),
which is also known as the cAMP response protein (CRP).
❑Within the lac Operon, CAP binds to an inverted repeat that is
centered near position -61 relative to the start site for transcription
17-Jan-21 20

21. 17-Jan-21 21

22. ❑The CAP-cAMP complex
stimulates the initiation of
transcription by approximately a
factor of 50.
❑A major factor in this stimulation
is the recruitment of RNA
polymerase to promoters to which
CAP is bound.
c) Positive control- CAP-cAMP binding
17-Jan-21 22

23. ❑An increase in the cAMP level inside an E. coli bacterium results in the
formation of CAP-cAMP complexes that bind to many promoters and
stimulate the transcription of genes encoding a variety of catabolic
enzymes.
❑Thus, the CAP-cAMP regulation acts as a positive regulator because its
presence is required for gene expression.
c) Positive control- CAP-cAMP binding
17-Jan-21 23

24. State of Lac Operon in the presence of only glucose
❑When grown on glucose, E. coli have a very low level of catabolic
enzymes such as β-galactosidase.
❑ It would be wasteful to synthesize these enzymes when glucose is
abundant.
❑The inhibitory effect of glucose, called catabolite repression, is due
to the ability of glucose to lower the intracellular concentration of
cyclic AMP.
17-Jan-21 24

25. State of Lac Operon in the presence of only glucose
❑The bacterium accumulates cAMP only when it is starved for a source
of carbon.
❑ In the presence of glucose—or of glycerol in concentrations sufficient
for growth—the bacteria will lack sufficient cAMP to bind to CAP because
the glucose inhibits adenylyl cyclase, the enzyme that converts ATP to
cAMP.
❑Thus, in the presence of glucose or glycerol, cAMP-saturated CAP is
lacking, so that the DNA-dependent RNA polymerase cannot initiate
transcription of the lac Operon.
17-Jan-21 25

26. 17-Jan-21 26

27. Lac I
Promoter
gene
Operator
gene
Lac Z Lac Y Lac A
mRNA
R R
RR
Lactose absentRepressor
molecules
Repressor
tetramer
No Gene
Expression
RNA polymerase
17-Jan-21 27

28. Lac I
Promoter
gene
Operator
gene
Lac Z Lac Y Lac A
mRNA
R R
RR
RNA polymerase
mRNA
Thiogalactoside
transacetylase
Permease
-
galactosidase Inducer
Inactive repressor
R
Lactose/
Isopropyl
Thiogalactoside
(IPTG) present
17-Jan-21 28

29. Glucose &
Lactose
present
Glucose 
cAMP level
No CAP-cAMP
complex
Promoter
not
identified
No
transcription
of ZYA genes
Lac Operon
repressed
No Gene
Expression
17-Jan-21 29
If there occurs no glucose metabolism

30. Lac I
Promoter
gene
Operator
gene
Lac Z Lac Y Lac A
cAMP 
Glucose pool gets depleted
due to metabolism
CAP-cAMP
complex formed
cAMP
CAP
RNA polymerase
mRNA
-galactosidase
Permease
Thiogalactoside
transacetylase
If there occurs glucose metabolism
R
IR
I
17-Jan-21 30

31. Summary- Regulation of Expression of Lac
Operon
1) In the absence of lactose- Lac Operon remains repressed due to the
presence of lac repressor at the operator site- (Negative control).
2) In the presence of only Lactose- Lac Operon is derepressed, the
structural genes are transcribed, and the lactose metabolizing
enzymes are synthesized (Double negative control).
17-Jan-21 31

32. Summary- Regulation of Expression of Lac Operon
3) In the presence of both glucose and lactose- CAP -cAMP
complex is not formed; RNA polymerase can not initiate the
transcription of structural genes even though the operator site is
vacant due to the binding of lactose/allolactose with lac
repressor.
Lac Operon remains in the repressed state. It is absence of
positive regulation.
17-Jan-21 32

34. Mechanism of regulation of gene expression
⚫Transcription control can result in tissue-specific gene
expression.
⚫In addition to transcription level controls, gene expression
can also be modulated by
⚫Gene rearrangement,
⚫Gene amplification,
⚫Post-transcriptional modifications, and
⚫RNA stabilization.
34

35. 1) Larger genome
• In comparison, the genome within a human cell contains 23 pairs of
chromosomes.
• Approximately 40,000 genes are present within the 3000 Mb of human
DNA.
• It would be very difficult for a DNA-binding protein to recognize a unique
site in this vast array of DNA sequences.
• More-elaborate mechanisms are required to achieve specificity
35

36. 2) Different cell types
⚫Different cell types are present in most
eukaryotes.
⚫Liver and pancreatic cells, for example, differ
dramatically in the genes that are highly
expressed.
⚫Different mechanisms are involved in the
regulation of such genes.
36

37. 3) Absence of operons
⚫The eukaryotic genes are not generally organized into operons as are
there in prokaryotes
⚫Instead, genes that encode proteins for steps within a given pathway are
often spread widely across the genome.
37

38. 4) Chromatin structure
⚫The DNA in eukaryotic cells is extensively folded
and packed into the protein-DNA complex called
chromatin.
⚫Histones are an important part of this complex
since they both form the structures known as
nucleosomes and also contribute significantly
into gene regulatory mechanisms.
38

39. 5) Uncoupled transcription and translation processes
• In prokaryotes, transcription and translation are coupled processes, the
primary transcript is immediately translated.
• The transcription and translation are uncoupled in eukaryotes, eliminating
some potential gene-regulatory mechanisms.
• The primary transcript in eukaryotes undergoes modifications to become
a mature functional m RNA.
39

40. Transcription and Translation in Prokaryotes and
Eukaryotes

41. 1) Chromatin Remodeling
• Chromatin structure provides an important level of
control of gene transcription.
• With few exceptions, each cell contains the same
complement of genes (antibody-producing cells are a
notable exception).
41

42. Chromatin Remodeling
• The development of specialized organs, tissues, and cells and
their function in the intact organism depend upon the
differential expression of genes.
• Some of this differential expression is achieved by having
different regions of chromatin available for transcription in cells
from various tissues.

43. Chromatin
Remodeling
⚫Large regions of chromatin are transcriptionally inactive in
some cells while they are either active or potentially active in
other specialized cells
⚫For example, the DNA containing the Beta-globin gene cluster is
in "active" chromatin in the reticulocytes but in "inactive"
chromatin in muscle cells.
43

44. Formation and disruption of nucleosome structure
• The presence of nucleosomes and of complexes of histones and
DNA provide a barrier against the ready association of
transcription factors with specific DNA regions.
• The disruption of nucleosome structure is therefore an
important part of eukaryotic gene regulation.
44

45. i) Histone Acetylation and deacetylation
45

46. Formation and disruption of nucleosome structure
ii) Modification of DNA
❑ Methylation of deoxycytidine residues in DNA may affect
gross changes in chromatin so as to preclude its active
transcription.
46

47. Formation and disruption of nucleosome structure
iii) DNA binding proteins
❑ The binding of specific transcription
factors to certain DNA elements may
result in disruption of nucleosomal
structure.
❑ Many eukaryotic genes have multiple
protein-binding DNA elements.
47

49. 2) Enhancers and Repressors
• Enhancer elements are DNA sequences, although they have no
promoter activity of their own but they greatly increase the
activities of many promoters in eukaryotes.
• Enhancers function by serving as binding sites for specific
regulatory proteins.
• An enhancer is effective only in the specific cell types in which
appropriate regulatory proteins are expressed.
49

50. 2) Enhancers and Repressors (contd.)
50

51. Activators and repressors

52. 3) Locus control regions
• Some regions are controlled by complex DNA elements called locus
control regions (LCRs).
• An LCR—with associated bound proteins—controls the expression
of a cluster of genes. The best-defined LCR regulates expression of
the globin gene family over a large region of DNA.
52
https://www.youtube.com/watch?v=vhB0oNLYIqo&t=4s

53. 3) Locus control regions and Insulators
53

54. 4) Gene Amplification
⚫The gene product can be increased by
increasing the number of genes available for
transcription of specific molecules
⚫Among the repetitive DNA sequences are
hundreds of copies of ribosomal RNA genes
and tRNA genes.
54

55. 5) Gene Rearrangement (contd.)
These DNA coding changes are
needed for generating the
required recognition diversity
central to appropriate immune
function.
55

56. 6) Alternative RNA Processing
⚫Eukaryotic cells also employ
alternative RNA processing to
control gene expression.
⚫This can result when alternative
promoters, intron-exon splice
sites, or polyadenylation sites
are used.
56

57. 6) Alternative RNA Processing (contd.)
⚫Alternative polyadenylation sites in the
immunoglobulin (Ig M) heavy chain primary
transcript result in mRNAs that are either
2700 bases long (m) or 2400 bases long (s).
⚫This results in a different carboxyl terminal
region of the encoded proteins such that
the (m ) protein remains attached to the
membrane of the B lymphocyte and
the (s) immunoglobulin is secreted.
57

58. 7) Class switching
❑ In this process one gene is
switched off and a closely related
gene takes up the function.
❑ During intrauterine life embryonic
Hb is the first Hb to be formed.
58

59. 7) Class switching (contd.)
⚫Gene switching is also observed
in the formation of
immunoglobulins.
⚫Ig M is the formed during
primary immune response, while
Ig G is formed during secondary
immune response.
59

60. 8) mRNA stability
⚫Although most mRNAs in mammalian cells are very stable (half-
lives measured in hours), some turn over very rapidly (half-lives
of 10–30 minutes).
⚫In certain instances, mRNA stability is subject to regulation.
⚫This has important implications since there is usually a direct
relationship between mRNA amount and the translation of that
mRNA into its cognate protein.
60

61. 8) mRNA stability (contd.)
⚫The stability of the mRNA can be influenced by hormones and certain
other effectors.
⚫The 5' cap structure in eukaryotic mRNA prevents attack by 5'
exonucleases, and the poly(A) tail prohibits the action of 3' exonucleases.
61

62. 9)DNA binding proteins
⚫Steroids such as estrogens bind to eukaryotic transcription
factors called nuclear hormone receptors. These proteins are
capable of binding DNA whether or not ligands are bound.
⚫The binding of ligands induces a conformational change that
allows the recruitment of additional proteins called co
activators.
62

63. 10) Specific motifs of regulatory proteins
⚫Three unique motifs—the helix-turn-helix, the zinc finger, and
the leucine zipper—account for many of these specific protein-
DNA interactions.
⚫The motifs found in these proteins are unique; their presence in
a protein of unknown function suggests that the protein may
bind to DNA.
⚫The protein-DNA interactions are maintained by hydrogen bonds
and van der Waals forces.
63

64. Three unique motifs of DNA binding proteins
Helix –turn- helix
Leucine zipper
Zinc finger
64

65. 11) RNA Editing
⚫Enzyme- catalyzed deamination of a specific cytidine residue in the mRNA
of apolipoprotein B-100 changes a codon for glutamine (CAA) to a stop
codon (UAA).
⚫Apolipoprotein B-48, a truncated version of the protein lacking the LDL
receptor-binding domain, is generated by this posttranscriptional change
in the mRNA sequence.
65

66. 11)RNA Editing (contd.)
66

68. Cloning
The procedure of cloning involves:
1) Cutting DNA at precise locations
2) Joining two DNA fragments covalently
3) Selection of a small molecule of DNA capable of self- replication
4) Moving recombinant molecules from the test tube in to a host cell
5) Selecting or identifying those cells that contain Recombinant DNA

69. 1) Cleavage of DNA
• Restriction Enzymes cut DNA Chains at specific locations
• The specific site is called “Restriction site” which is 4-7 base pair long
• The fragments of DNA obtained after the action of restriction enzymes
are called “Restriction fragments”; they can have sticky or blunt ends.
• Restriction enzymes are also called “Molecular scissors”

70. 2) Joining two DNA fragments covalently
The DNA fragments are
joined together by DNA ligase

71. 3) Selection of a small molecule of DNA capable of self- replication
Chimeric or hybrid DNA molecules can be constructed in cloning vectors
which then continue to replicate in a host cell under their own control
systems. In this way, the chimeric DNA is amplified.
Cloning vectors
A vector is a molecule of DNA to which the fragments of DNA to be cloned is
attached.

72. Cloning vectors
Commonly used vectors
• Plasmids
• Bacterial and animal viruses
• Cosmids
• Artificial chromosomes

73. Bacterial Plasmids
● Bacterial plasmids are small, circular, duplex DNA molecules whose
natural function is to confer antibiotic resistance to the host cell.
● Plasmids have several properties that make them extremely useful as
cloning vectors.

74. Bacterial and animal viruses
● Phages usually have linear DNA molecules
into which foreign DNA can be inserted at
several restriction enzyme sites.
● A major advantage of phage vectors is that
while plasmids accept DNA pieces about
6–10 kb long, phages can accept longer
DNA fragments.

75. Cosmids
Larger fragments of DNA can be cloned in
cosmids, which combine the best features
of plasmids and phages.

76. Artificial Chromosomes
Even larger pieces of DNA can be incorporated
into bacterial artificial chromosome (BAC) or
yeast artificial chromosome (YAC).
These vectors can accept and propagate DNA
inserts of several hundred kilobases or more
and have largely replaced the plasmid, phage,
and cosmid vectors.

77. Viral vectors
Mammalian viral vectors are also
used in this technology especially
for gene therapy.

78. 4) Moving recombinant molecules from the test tube into a host cell
E. coli is the most common host cell due to the
following advantages:
i) It’s DNA metabolism is well understood
ii) Cloning vectors associated with E.Coli are
well characterized
iii) Effective techniques are available for
moving DNA from one bacterial cell to
another

79. Transformation
● The phenomenon of transformation permits plasmid vectors to be
introduced into and expressed by E. coli cells.
● Normal E. coli cells cannot take up plasmid DNA from the medium.
● Exposure of cells to high concentrations of certain divalent cations,
however, makes a small fraction of cells permeable to foreign DNA by a
mechanism that is not understood.

80. Transformation
● In a typical procedure, E. coli cells are
treated with CaCl2 and mixed with
plasmid vectors;
● commonly, only 1 cell in about 10,000 or
more cells becomes competent to take up
the foreign DNA.
● Transformation can also be undertaken by
giving a heat shock

81. 5) Selecting or identifying those cells that contain Recombinant DNA
● Colony or plaque hybridization is the
method by which specific clones are
identified and purified.
● Bacteria are grown as colonies on an agar
plate and overlaid with nitrocellulose
filter paper.
● Cells from each colony stick to the filter
and are permanently fixed thereto by
heat, which with NaOH treatment also
lyses the cells and denatures the DNA so
that it will hybridize with the probe.

82. Selecting or identifying those cells that contain Recombinant DNA
● A radioactive probe is added to the filter, and
(after washing) the hybrid complex is localized
by exposing the filter to x-ray film.
● By matching the spot on the autoradiograph to
a colony, the latter can be picked from the
plate.
● A similar strategy is used to identify fragments
in phage libraries.
● Successive rounds of this procedure result in a
clonal isolate (bacterial colony) or individual
phage plaque.

83. Colony or plaque hybridization

84. DNA libraries
● The combination of restriction enzymes and various cloning vectors
allows the entire genome of an organism to be packed into a vector.
● A collection of these different recombinant clones is called a library. A
genomic library is prepared from the total DNA of a cell line or tissue.
● A cDNA library comprises complementary DNA copies of the population
of mRNAs in a tissue.
● A cDNA library comprises complementary DNA copies of the population
of mRNAs in a tissue.
● It represents a collection of only the genes that are encoded into
proteins by an organism.

85. Genomic library cDNA library

86. DNA probes
● A variety of molecules can be used to "probe"
libraries in search of a specific gene or cDNA
molecule or to define and quantitate DNA or
RNA separated by electrophoresis through
various gels.
● Probes are generally pieces of DNA or RNA
labeled with a 32P-containing nucleotide—or
fluorescently labeled nucleotides (more
commonly now).
● Biotinylated probes are also in practice.

87. Expression vectors
These vectors are specially constructed to
contain:
● very active inducible promoters, proper
in-phase translation initiation
codons,both transcription and
translation termination signals, and
appropriate protein processing signals,
if needed.
● Some expression vectors even contain
genes that code for protease inhibitors,
so that the final yield of product is
enhanced.

88. Southern hybridization
It is named for the person
who devised the technique
[Edward Southern], and the
other names began as
laboratory jargon but are
now accepted terms

89. Restriction fragment length polymorphism
● Treatment of genomic DNA from different individuals with a
single restriction enzyme does not always give the same set of
fragments
● because some restriction sites are polymorphic,
● being present in some individuals but absent in others,
● usually because a point change in the nucleotide sequence
changes the restriction site into a sequence not recognized by
the restriction enzyme

90. Restriction fragment length polymorphism

91. Applications of Southern blot
DNA fingerprinting is an example of southern blotting.
● Used for paternity testing, criminal identification, and victim
identification.
● To identify mutation or gene rearrangement in the sequence of DNA.
● Used in diagnosis of diseases caused by genetic defects.
● Used to identify infectious agents.

92. Northern blotting

93. Western Blotting

95. Overview of Applications of r-DNA technology in the field of
medicine
● Mapping out the human genome and the creation of transgenic animals,
● Genetic testing in forensic science and archaeology
● Tests for determining hereditary disease and paternity.
● Diagnostic tests for hepatitis and human immunodeficiency virus (HIV).
● Production of vaccines and protein therapies such as human insulin,
interferon and human growth hormone.
● Production of clotting factors for treating haemophilia and in the
development of gene therapy.

96. Genetic mapping (linkage mapping)
“Gene mapping is a process or method of discovering the location of genes
on a chromosome.” It helps to identify:
● that a disease transmitted from parent to child is linked to one or more
genes.
● which chromosome contains the gene and precisely where the gene lies
on that chromosome.
● the gene responsible for relatively rare, single-gene inherited disorders
such as cystic fibrosis and Duchenne muscular dystrophy.
● the genes that are believed to play a role in the development of
common disorders such as asthma, heart disease, diabetes, cancer, and
psychiatric conditions.

97. Gene therapy
● Diseases caused by deficiency of a gene product are
amenable to replacement therapy.
● The strategy is to clone a gene into a vector that will
readily be taken up and incorporated into genome of a
host cell.
● Adenosine deaminase deficiency has been treated
successfully with gene replacement therapy.
● For many sickle cell anaemia, thalassaemias, and
various other metabolic disorders Gene therapy is
under trial.

98. Transgenesis
The somatic gene replacement therapy can not
pass on to the offspring. Transgenesis refers to
the transfer of genes into fertilised ovum which
can be found in somatic as well as germ cells
and passed on to the successive generations.
Injection of the gene for growth
hormone into a fertilized mouse egg
gave rise to a giant
mouse (left), about twice the weight
of his sibling (right).

99. Manufacture of proteins/hormones
● By inserting the gene for a rare protein into a plasmid and expressing it in
bacteria, large amounts of the recombinant protein can be produced. Many
proteins have been produced e.g., human growth hormone, insulin,
interferons and blood clotting factors.
● Another approach to producing proteins via recombinant DNA technology is
to introduce the desired gene into the genome of an animal, engineered in
such a way that the protein is secreted in the animal’s milk, facilitating
harvesting.

100. Manufacture of proteins/hormones

101. Diagnosis of Infectious disease
Diagnosis of HIV infection- The widely used methods for diagnosing HIV
infection have been developed using recombinant DNA.
● The antibody test (ELISA or western blot) uses a recombinant HIV
protein to test for the presence of antibodies.
● The DNA test detects the presence of HIV genetic material using reverse
transcriptase polymerase chain reaction (RT PCR).
● Development of the RT-PCR test was made possible by the molecular
cloning and sequence analysis of HIV genomes.

102. Diagnosis of molecular diseases
● Many genetic diseases that yield developmental abnormalities can be
detected by characteristic patterns in DNA primary structure.
● Such mutational changes in DNA sequences are identified by restriction
fragments analysis and Southern blotting, using appropriate DNA
probes.
● Analysis of this type could be done in understanding the molecular basis
of diseases like sickle cell anaemia, thalassaemias, familial
hypercholesterolaemia, cystic fibrosis, etc.

103. Prenatal diagnosis
● In diseases where the genetic defect is
known and a specific probe is available,
prenatal diagnosis can be made.
● DNA from cells collected from as little as
10 ml of amniotic fluid or by chorionic
villi biopsy can be analysed by Southern
blot transfer.

104. Application in forensic medicine:
Advances in genetic engineering have greatly
helped to specifically identify criminals and
settle the disputes of parenthood of children.
Based on the basis of Restriction fragment
length polymorphism, the identity of a
person can be confirmed.

105. Applications in agriculture:
● Genetically engineered plants have been
developed to resist drought and diseases.
● Good quality of food and increased yield of
crops can be possible by applying this
technology.
● Incorporation of nif genes to cereals has
given higher yield of the crops.

106. Industrial applications
● Enzymes synthesised by this technology are used to produce sugars,
cheese and detergents.
● Certain protein products produced by this technology are used as food
additives to increase the nutritive value, besides imparting flavour.
● Ethylene glycol is in great demand for industry.
● Preparation of ethylene glycol from ethylene is made possible by this
technology.

107. Animal cloning

108. Biowar

109. rDNAT
Medicine
Diagnosis
Infectious
diseases
Genetic
disorders
Identity
ConfirmationPrevention
Vaccines
Treatment
Drugs
Gene
therapy
Animal
cloning
Agriculture
Quality
Quantity
Protection
Industries
Sugars, cheese,
detergentsBio-war
Gene
mapping
Transgenesis

111. Polymerase chain reaction (PCR)
• A molecular technique to copy or amplify small segments of DNA or
RNA.
• An efficient and cost-effective technique that combines the principles of
complementary nucleic acid hybridization with those of nucleic acid
replication that are applied repeatedly through numerous cycles.
• It results in the exponential production of the specific target DNA/RNA
sequences by a factor of 107 within a relatively short period.

112. Components of Polymerase Chain Reactions (PCR)
DNA template (the sample DNA that contains the target sequence to
amplify)
• Deoxyribonucleoside triphosphates (dNTPs)
• PCR buffer
• Primers (forward and reverse)
• Taq polymerase

113. Steps of procedure
• The PCR is carried out in a single test tube containing all the necessary
components.
• The extracted sample (which contains target DNA template) is added to
the tube containing primers, free nucleotides (dNTPs), and Taq
polymerase.
• The PCR mixture is placed in a PCR machine, that increases and
decreases the temperature of the PCR mixture in automatic,
programmed steps and the copies of the target sequence are generated
exponentially.

114. Steps of procedure
• The PCR amplification occurs by
repeated cycles of three
temperature-dependent steps
called:
• denaturation,
• annealing, and
• elongation

115. Steps of procedure
1) Denaturation (strand separation):
• Native DNA exists as a double helix
• Denaturation separates the two DNA chains by
• heating the reaction mixture to 90°C to 95°C (194°F to 203°F).

116. Steps of procedure
2) Annealing (primer binding):
• the reaction mixture is cooled to 45°C to 60°C (113°F to 140°F)
• so that the oligonucleotide primers can bind or anneal to the separated
strands of the target DNA.

117. Steps of procedure
3) Extension (synthesis of new DNA):
During elongation, the DNA polymerase adds nucleotides to the 3 ‘ends of
the primers to complete a copy of the target DNA template.
These three steps are repeated 20-30 times in an automated thermocycler
that can heat and cool the reaction mixture in tube within very short time.
This results in exponential accumulation of specific DNA fragments.

118. Steps of procedure
• PCR is a method to amplify any DNA sequence
virtually without limit and allows the separation of
the nucleic acid of interest from its context.
• The doubling of number of DNA strands
corresponding to target sequences can be
estimated by amplification number associated
with each cycle using the formula.
• Amplification=2n, where n=no. of PCR cycle

119. PCR
• PCR can amplify a desired DNA sequences of any origin hundred or
millions time in a matter of hour, which is very short in comparison to
recombinant DNA technology.
• PCR is especially valuable because the reaction is highly specific, easily
automated and very sensitive.
• It is widely used in the fields like- clinical medicine for medical diagnosis,
diagnosis of genetic diseases, forensic science; DNA fingerprinting,
evolutionary biology.

120. Steps of
procedure

121. Detection of PCR products
Probe based detection of amplicons serves two purposes:
1.It allows visualization of the PCR product
2.It provides specificity by ensuring that the amplicon is the target
sequence of interest and not the result of non-specific amplification.
Apart from DNA based hybridization method, sometimes simple gel
electrophoresis method is sufficient to confirm the presence of specific
amplicons.

122. Applications of PCR
1.Forensic science: DNA fingerprinting, paternity testing and criminal
identification
2.Diagnosis: Identification and characterization of infectious agents
• Direct detection of microorganisms in patient specimens
• Identification of microorganisms grown in culture
• Detection of antimicrobial resistance
• Investigation of strain relatedness of pathogen of interest

123. Applications of PCR
3. Detection of mutation (investigations of genetic diseases)
4. Evolution study: evolutionary biology
5. Fossil study: paleontology
6. Gene cloning and expression
7. Gene sequencing
8. Vaccine production by recombinant DNA technology
9. Drug discovery
10. Human genome project

124. Question
A 10-month-old baby boy presents with steatorrhea, recurrent
pulmonary infections, GI upset and foul-smelling stool. Which of the
following tests is undertaken to confirm your diagnosis?
a) Northern hybridization
b) Western hybridization
c) ELISA
d) RFLP analysis

125. Answer
d) RFLP analysis

126. Question
Which of the following techniques is primarily undertaken to amplify
the DNA?
a) PCR
b) Microarrays
c) Northern Blotting
d) Southern Blotting

127. Answer
a) PCR

128. Question
All the following are used in PCR EXCEPT:
a) Taq polymerase
b) Restriction enzymes
c) Oligonucleotide primers
d) Deoxyribonucleoside triphosphates

129. Answer
b) Restriction enzymes

130. Question
Which out of the following mechanisms is involved in the production of
variety of immunoglobulins each specific for a specific antigen?
a) Class switching
b) Gene amplification
c) Gene rearrangement
d) RNA editing

131. Answer
c) Gene rearrangement

132. Question
Which of the following does not have introns?
a) DNA
b) Non-processed pseudo genes
c) Processed m RNA
d) Primary RNA transcript

133. Answer
c) Processed m RNA

134. Question
With respect to the LAC operon, if both glucose and lactose are present
and glucose is low, which of the following is NOT true?
a) High CAP
b) increased uptake of lactose
c) low cAMP
d) increased transcription of the lac operon

135. Answer
c) low cAMP

136. Question
Northern blotting is used for separation of:
a) DNA
b) mRNA
c) Protein
d) Protein DNA interactions

137. Answer
b) mRNA

138. Question
Which out of the following techniques is used for the detection of gene
of interest –
a) Southern Blotting
b) Polymerase chain reaction
c) Northern Blotting
d) DNA Foot printing

139. Answer
a) Southern Blotting

140. Question
Which histone is NOT part of the nucleosome?
a) H1
b) H2A
c) H2B
d) H3

141. Answer
a) H1

142. Question
Alternative splicing…
a) Creates protein from multiple segments of DNA on different
chromosomes
b) Is the reason why the human genome is much more complex than
other species
c) Creates different proteins from a single gene
d) is not tissue specific

143. Answer
c) Creates different proteins from a single gene

144. Question
DNA methylation is associated with:
a) CpG islands
b) CAT box
c) TATA box
d) increasing gene transcription

145. Answer
a) CpG islands

146. Thank you