Bacterial genetics. Basics

1. Bacterial Genetics
Learning the Basics
Dr.T.V.Rao MD
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2. Genetics Guide Life
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3. Genes are Eternal
Run In Progeny
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4. Understanding Genetics
We resemble and differ because of
Genetic configurations
Parents - Son - Daughter, how they
resemble each other.
They breed true from Generation to
Generation
But vary in small proportions in
progeny.
Bacteria too obey the laws of
Genetics Dr.T.V.Rao MD 4

5. Watson - Crick
Discovery of DNA
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6. Beginning of Bacterial
Genetics
• The principles of Genetics were applied to
bacteria and viruses
• Advances in Genetic process also of lead
to fundamental advances in Biology and
Biochemistry.
A Birth of New Branch of Science
Molecular Biology
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7. DNA
A Complex Structure
Makes Life
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8. DNA
( Deoxyribonucleic Acid )
• DNA is composed of Many Units of
Adenine – Thymine A – T
Guanine – Cytosine G - C
A+ T
G+C proportion differ for each species
DNA replicates first unwinding at one end to form a
fork
Each strand of fork acting as template for the
synthesis of complementary strand
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10. Structure of DNA
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11. DNA
• A DNA molecule is
composed of two
chains of Nucleotides
wound together in the
form of a Double
Helix
• Each chain has back
bone of Deoxyribose
and Phosphates
residues arranged
alternatively Dr.T.V.Rao MD 11

12. Structure of DNA
• Attached to each
Deoxyribose and
phosphate residues
arranged alternatively
• Attached to each
Deoxyribose are of four
nitrogen bases
• Purines - Adenine,
Guanine
• Pyramidine
Thymidine and Cytosine
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13. How RNA differs from DNA
• RNA contains - Sugar Ribose instead
of Deoxyribose
• Uracil is present instead of Thymine
• Types of RNA
Messenger RNA mRNA
Ribosomal RNA rRNA
Transfer RNA tRNA
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14. DNA - RNA
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15. Knowledge on DNA lead to
advances in Molecular Biology
• Central dogma of Life – Deoxyribonucleic acid
• DNA carries the Genetic information
• DNA is transcribed to RNA – Polypeptides
Cell Function depends upon specific
polypeptides – Proteins – Enzymes
DNA is a store house of Protein synthesis
DNA acts a Template for synthesis of mRNA
Virus differs from other as they contains either
DNA or RNA
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16. What is a Code in Genetics
• Code is a unit consists of sequence of three
Bases
• Code is triplet A-T- C
• A code can make single Amino acid
• More than one code present for making similar
sequence of Amino acid
• AGA make Arginine
• AGC, CGU, CGG, also code for similar Amino
acid
• Some Codons UAA Dr.T.V.Rao MD
don't code for any Amino 16
acid called as Nonsense codon

17. What is a Gene
• Gene is a sequence
of DNA carrying
codons specifying for
particular polypeptide.
• DNA contains many
Genes( A
combinations of
hundreds and
thousands of
Nucleotides )
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18. Bacterial Chromosome
• Contains a Double stranded molecules of
DNA arranged in circular form.
• Length 1,ooo microns.
• Bacterial DNA contains about
4,000kilobases
• I kb = 1000 base pairs ( A-T ) ( G-C)
• Humans have about 3,000 kb pairs.
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19. How bacterial Genome differs
from Higher forms of Life
• Several stretches of DNA don't appear to
function as codons, occurs between the
coding sequences of Gene. called as
INTRONS.
• Coded are called as EXONS
• In transcription introns are excised when
form RNA before translated by ribosomal
proteins.
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20. Extra chromosomal
Genetic Elements
Bacteria posses Extra chromosomal
genetic elements
Not Essential for survival of Bacteria
But makes the Bacteria Resistant to
antibiotics, and makes them survive
Able to produce toxins
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21. Plasmids
• Plasmids are circular
DNA molecules present
in the cytoplasm of the
Bacteria
• Capable of Autonomous
replication
• Can transfer genes from
one cell to other
• Act as vectors in Genetic
engineering.
• Can also present in
Yeasts
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22. Plasmid ( Blue )
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23. Plasmids
• Plasmid seem to be ubiquitous in bacteria, May
encode genetic information for properties
1 Resistance to Antibiotics
2 Bacteriocins production
3 Enterotoxin production
4 Enhanced pathogen city
5 Reduced Sensitivity to
mutagens
6 Degrade complex organic molecules
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24. R plasmid
R: drug resistance
RTF: transfer of R
plasmid
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25. Plasmids
• Can be integrated
with Chromosomal
DNA
• Episomes -
Integrated form of
plasmid with DNA
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26. Potentials of Plasmids
• Plasmids can be self
transmissible and
Non transmissible
• Transfers the Sex and
Drug resistance with
the help of restriction
end nucleases
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27. Classification of Plasmids
• Incompatibility typing
• Don't accommodate
others which are
similar
• Other methods of
Classification
Centrifugation
Electrophoresis
Genetic methods
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28. Genotypic and Phenotypic
variation
• Genome – Sum total of Gene that make
up the genetic apparatus of cell
established as Genotype.
• Hereditary constitution of cell this
transmitted to its progeny
• Phenotype – is the physical expression in
a environment. Change according to
environment.
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29. What is Phenotypic expression
• Exhibit – different phenotypes
• Appearance differs in different situations.
• Eg Typhoid bacilli flagellated normally
• But grown in Phenol agar don't grow flagella So
flagella are lost physical variation
• Lactose fermentation in E.coli dependent on
Beta Galactosidase
When lactose present - test is positive
When lactose is absent - test turns negative
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30. Different Enzymes Guided by
Genomic configurations
• Inducer enzyme acts in the presence
of substrate
• Constitutive enzyme acts irrespective
of presence or absence of enzyme.
• Phenotypic variations influenced by
environment limited in range by
genes
• Genotypic variations are stable not
influenced by environment.
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31. Principles of Genotypic
variations
• Mutations
• Genotypic by transfer of genes
Transformation
Transduction
(Lysogenic conversion)
Conjugation
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32. Replica Plating, pt. 2
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33. Mutations in Bacteria
• Bacteria Multiply by asexual binary fission
• Altered Nucleotide sequence in expresses new
or altered characteristics
• Selective value to the organism
• Evolutionary value
• Acquires Antibiotic resistance grows in body
without inhibition
• Become a prominent organism
• Phenotypic variation occurs when genes
changes in response to the environment but
reversible.
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34. Mutations
• Mutation is a Random, Undirected,
Heritable variation
• Caused by alteration in the Nucleotide
sequence at some point of DNA which can
occur due to
Addition
Deletion
Substitution
of one or more bases
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35. Mutation Type
Frameshift (deletion)
(leu) (ser) (arg)
Normal AAT AGT GCC
(leu) (val) (pro)
Mutant AAT AGT GCC A
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36. Mutation Type
Frameshift (insertion)
(leu) (ser) (arg)
Normal AAT AGT GCC
(leu) (glut) (cyst)
Mutant AAT CAGT GCC
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38. Mutations can occur in any
sequence,inveitable, useful for
Survival
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39. Multiple Mutations
• Causes extensive chromosomal rearrangement
• Missense mutation -Triplet code is acted so as
to specify an Aminoacid different from that
normally located at particular position in the
protein
• Nonsense mutation - Deletion of nucleotide
within a gene may cause premature polypeptide
chain termination by nonsense codon
• Tran version is Substitution of purine for
pyramidine or vice versa in the base pairing
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40. Mutations
• Suppressor Mutation is reversal of mutant
phenotype by another mutation at a point of
DNA distant from that of original mutation.
• All genes are susceptible for mutations, but all
mutations are not expressed
• Lethal mutation is harmful destroy the vital
functions
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41. Mutations
• Conditional Lethal mutant may be Live under
certain conditions
• Common example is temperature ( its ) mutant
• Temp sensitive ( ts) mutant lives at 350c but not
at 390c
• Each gene undergoes mutation at a fixed
frequency.
Bacteria undergo mutations at 10-4 - to 10-10
• Tutomerisim T – A is replaced by G - A
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42. Mutagenic Agents
• U V rays
• Alkyl ting
agents
• Arcidine Dyes
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43. GENE TRANSFER
Occurs by Complex Mechanisms
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44. The three bacterial sexual
processes
– 1.Conjugation: direct transfer of
DNA from one bacterial cell to
another.
–2. Transduction: use of a
Bacteriophages (bacterial virus) to
transfer DNA between cells.
–3. Transformation: naked DNA is
taken up from the environment by
Dr.T.V.Rao MD 44
bacterial cells.

45. Transformation of Genetic
material
( Gene Transfer )
• Different
Mechanisms
Transformation
Transduction
Conjugation
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46. Gene Transfer Processes for
Bacteria and Their Viruses
1. Conjugation
2. Transformation
3. Transduction
4. Infection with
bacteriophage
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47. What is Transformation
• Transformation is defined as transfer of Genetic
information through the activity of DNA
• Griffith experiment
Mice injected with Live non capsulated ( R )
Pneumococci
with heat killed capsulated (S) Pneumococci
Lead to death of Mice with isolation of Live
capsulated Pneumococci
It means that some factor from Dead
pneumococci transferred to live non pathogenic
Pneumococci
Avery, McCleod, Mc Cartny in 1944 identified to be DNA
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48. Griffith Phenomenon
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49. Demonstration of
transformation
Avery, MacLeod, and
McCarty (1944)
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50. Transduction
• Generalized involve any segment of DNA
• Restricted when specific Bacteriophages
traduces only a particular genetic trait.
• Transduction effects Plasmids ,and Episomes
• Plasmid transfer induces Penicillin resistance in
Staphylococcus
• Helps Genetic mapping, also in eukaryotic cell
• Helps Genetic Engineering
T.V.Rao MD
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51. Transduction
• Transduction is
defined as
transfer of
portion of DNA
from one
bacteria to
another by
Bacteriophages,
is known as
Transduction Dr.T.V.Rao MD 51

52. DNA transfer through
Bacteriophages
• When the Phage
particle infects
another bacteria
DNA transfer is
effected and the
recipient cell
acquires new
characters coded
by donor DNA
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53. Bacteriophages
• Are viruses that
parasitize bacteria
and consists of
Nucleic acid core and
a protein coat
• A phage particle may
have at its core
besides its own
nucleic acid and a
segment of the Host
DNA Dr.T.V.Rao MD 53

54. Transduction Types
• Two types of Transduction
• 1 Lytic and 2 Lysogenic
• 1 Virulent or Lytic cycle after
large number of progeny are
built up inside the host
bacterium ruptures and
phages are released
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55. Lysogenicity creates new
characters
• Eg - Lysogenic conversion in
Diphtheria bacilli which
acquires toxigenicity by
lysogenization with phage beta
• Elimination of phage for
toxigenic strain renders
nontoxigenic
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56. Conjugation
Lederberg - Tatum
• A process by which a Donor cell or male cell
makes contact with another cell, the recipient or
Female cell.
• DNA is directly transferable
• Plasmid Carry genetic information necessary for
conjugation to occur.
• Only cell that contain such plasmids can act as
donor. the cell lacking a corresponding plasmid
act as recipient.
• Requires direct contact between donor and
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recipient

57. Conjugation
• The ability to conjugate is
conferred by the F plasmid. A
plasmid is a small circle of DNA
that replicates independently of
the chromosome. Bacterial cells
that contain an F plasmid are
called “F+”. Bacteria that don’t
have an F plasmid are called “
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58. Conjugation - Transferring
genes with plasmids
• Plasmids
mediating
conjugation carry
genes coding for
properties, of 1-2
microns long
protein appendage
termed Pilus on the
Donor cell
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59. Mechanism of Transfer I:
Conjugation
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60. Conjugation
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61. Simple Conjugation
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62. Conjugation
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63. Pilus helps Conjugation
• Different types of Pilus
are specified by different
types of plasmids and
can help in aid of plasmid
classification.
• Only one strand of
circular DNA of the
plasmid nicked upon at a
specific site and passed
into a recipient.
• Spread to all other cells.
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64. F factor
• Transfer factor that
contains the genetic
information necessary
for synthesis of Sex
Pilus and for self
transfer without any
other identifiable
genetic materials
such as drug
resistance
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65. F factor helps transformation
• F+ called as Donor bacteria can transform
F- into F+ cell
Can be Episomes able to exist in some cells in the
integrated state in the donor cell chromosome
Can transform chromosomal genes to recruitment with
high frequency are known as Hfr cells
Conversion of F+ cells into Hfr state is reversible.
F factor incorporates some chromosomal genes and is
called as F’
Sexduction The process of transfer of host genes
through F’ factor
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66. DNA transfer through
Bacteriophages
• When the Phage
particle infects
another bacteria
DNA transfer is
effected and the
recipient cell
acquires new
characters coded
by donor DNA
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67. Types of DNA transfer through
Bacteriophages
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68. Colicinogenic ( Col ) Factor
• Coli form Bacteria
produce Colicins
• Colicins are lethal to
other Enterobacteriaceae
• Pyocins produce by
Pseudomonas
• Diptherocins produced
by C.diptheria
• Plasmid transmits col factor
leads to self transfer of
chromosomal segments
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69. Resistance Transfer Factor
RTF
• Plasmids – helps to spread multiple drug
resistance
• Discovered in 1959 Japan
• Infections caused due to Shigella spread
resistance to following Antibiotics
Sulphonamides
Streptomycin
Chloramphenicol,
Tetracycline
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70. RTF
• Shigella + E.coli
excreted in the stool
resistant to several
drugs in vivo and vitro
• Plasmid mediated –
transmitted by
Conjugation
• Episomes spread the
resistance
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71. Bacterial Conjugation:
High Frequency Transfer (Her) Cells
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72. Hfr Conjugation
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73. Sequence of RTF transmission
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74. Hfr cell conjugating a Normal cell
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75. Composition of RTF
• Plasmid consists of two
components
• A transfer factor RT, helps
conjugational transfer and
resistant determinants ( r ) to
each of the several drugs
• RTF + r determinants are known
as R factor
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76. R factor
• R factor can contain
several determinants as
many as 8 or > 8 drugs
• Guide the cell for
production of
Enterotoxins too
• But R factors can be
inhibited by
Bile salts
R factors can be
transferred to animals
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77. Genesis of R factors
• In discriminate use of
Antibiotics in vet nary Medicine
has increased the spread of R
factors to Human
• Addition of Antibiotics to
Animal feeds to be prohibited.
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78. Genetic Mechanisms of Drug
Resistance
• Bacteria acquire drug resistance through several
Mechanisms
• Mutations
• Genetic transfer
Transformation,
Transduction
Conjugation
Several Biochemical Mechanisms
Decreasing permeability of drugs,
Attaining alternative pathways
Produce enzymes and inactivate drugs
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79. Genetic Mechanisms in Bacteria helps to
Spread the Infectious diseases
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80. Mutations
• Mutilations can be
1 Stepwise mutation as in Penicillin use
2 One step mutation
Streptomycin use
May show low resistance or High resistance
If tuberculosis is treated with sole drug as of Only
Streptomycin some resistant mutants appear
and replaces sensitive bacteria in due course
so the occurrence of MDR - TB
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81. Other Mechanisms
• Use of Penicillin created resistant
Staphylococcus by transduction
• R factors created resistance to several
drugs, caused increased virulence
• Spread to several humans and animals
Best option- To restrict use of
Antibiotics
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82. Transposable Genetic Elements
Structurally / Genetically – Discrete sequence of
DNA – Move around in a cut and paste manner
between Chromosomal and Extra chromosomal
DNA molecules within cells.
Called as Transposons _ Jumping Genes
Genetic transfer due to Transposition
Small Transposons 1 – 2 Kb
Not self replicating and depend on Plasmid or
Chromosome for replication.
A chunk of DNA is added by Transposons.
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83. Transposons and R factor
• R forms may have evolved as a collection of
Transposons
• Each carrying Genes that confers resistance to
one or several Antibiotics
• Seen in Plasmids,
Microorganisms
Animals
Laboratory Manipulations are called as Genetic
Engineering
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84. Molecular Genetics
• Analysis and
manipulation of
DNA using
Biochemical
and
Microbiological
techniques Dr.T.V.Rao MD 84

85. Genetic Engineering
• Under standing Molecular
genetics in Biochemistry
fuels genetic Engineering
• Recombinant DNA
(renal) techniques
changed the ideals of
Medicine
• Genetic Engineering
await many
surprises?
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86. Genetic Engineering
Genetic Engineering Was Born from
Genetic Recombination
•Genetic engineering involves changing
the genetic material in an organism to
alter its traits or products
•A recombinant DNA molecule contains
DNA fragments spliced together from 2 or
more organisms Dr.T.V.Rao MD 86

87. Modern applications
• Pharmaceutical production
–Insulin, interferon, hormones, vaccines
etc.
• Genetically engineered plants
• Animal gene alterations
• Gene probes
• DNA fingerprinting
• The human genome initiative
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88. Genetic Engineering
• Isolation of Genes coding for any
desired protein from Microorganism
or from cell of higher life forms
including human beings and their
introduction into a suitable
microorganism in which genes would
function directing the production of
specific proteins
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89. Genetic Engineering changing
the Diagnostic and Therapeutic
Protocols in
MEDICINE
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90. Research on Gene transfer
shapes the future of Science
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91. Genetically Engineered Products
• Can prepare desired
protein in pure form in
economic way
Somatostatin
• Commercial
preparations pdf
Cloned Human
Insulin
Interferons
Hepatitis B
vaccine Dr.T.V.Rao MD 91

92. Restriction Endonucleases
• A restriction enzyme (or restriction
endonuclease) is an enzyme that cuts
double-stranded DNA. The enzyme
makes two incisions, one through each of
the sugar-phosphate backbones (i.e., each
strand) of the double helix without
damaging the nitrogenous bases They
work with cutting up foreign DNA, a
process called
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93. Restriction Endonucleases
Made the advances in Genetic
Engineering
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94. DNA Probes
• There are Radioactive
Biotinylated otherwise
labeled copies united
single stranded DNA
Contains 20 -25 nucleotides
Helps detection of
Homology DNA by
Hybridization.
Helps Diagnosis of
Infectious Diseases
Minute quantities of DNA
can be detected.
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95. Blotting Techniques
• Drug fragments obtained by restriction
enzyme digestion on separation Gel can
be transferred to Nitrocellulose or nylon
membranes
• Several methods
1 Southern blotting
2 Northern Blotting
3 Western blotting
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96. western blot
• The western blot (alternatively, protein
immunoblot) is an analytical technique
used to detect specific proteins in a given
sample of tissue homogenate or extract. It
uses gel electrophoresis to separate
native or denatured proteins by the length
of the polypeptide (denaturing conditions)
or by the 3-D structure of the protein
(native/ non-denaturing conditions)
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97. Western Blotting
• In Western Blot Protein (
Antigen ) mixture is
separated by SDS (
Sodium dodecyl sulfate –
polyacrylamide gel
electrophoresis ) Blotted
on to Nitro cellulose strips
and identified by radio
labeled or enzyme
labeled antibodies as
probes
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98. Western Blot
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99. Western Blot to confirm
HIV Infections made land mark
Diagnostic tool
• Western Blot testing
is confirmatory test for
diagnosis of
HIV/AIDS
• Identifies antibodies
directed against
different antigens in
pathogen
Surface,
Core
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RT antigen

100. Polymerase chain reaction
Kary B Mullis 1983
• Rapid
• Automatic
amplification of
specific DNA
sequences
• Nobel prize winning
Technology 1993
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101. PCR -Sequences
• PCR consists of several
cycles of sequential DNA
replication where the
products of first cycle
becomes the template for
the Next
• It makes available
abundant quantities of
specific DNA sequences
starting
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102. Genetic Mapping
• Genetic sequences
for Bacteriophages
and virus
• Genetic mapping is
done most of the
Human Genes
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103. Newer Understanding on
GENES
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104. Human Genome Project
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105. Human Genome Project
• Completed in 2003, the Human Genome
Project (HGP) was a 13-year project
coordinated by the U.S. Department of
Energy and the National Institutes of
Health. During the early years of the HGP,
the Welcome Trust (U.K.) became a major
partner; additional contributions came from
Japan, France, Germany, China, and
others
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106. Genetics are Complex - Leading
the birth of BIOINFORMATICS
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107. Genes Evolved and made us
Men
What NEXT ?
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108. Are We Playing with Genes in the
Right Direction ?
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109. Understanding of human Genome is
Changing the Future of Medicine
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110. • Programmed Created by Dr.T.V.Rao MD
for Undergraduate Medical Students in
the Developing World
• Email
• doctortvrao@gmail.com
Dr.T.V.Rao MD 110