Most Popular tool of genetic

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Used to obtain structural
information of DNA fragment DNA Mapping/ Restriciton Mapping
DNA molecule to be sequenced is
digested with restriciton enzyme Gene Sequencing Applications
Gene expression & mutation
studies
Examination of population
polymorphisms

Bifunctional enzyme
Structure
Immunoblotting
Three different subunits Subunits
ATP, Mg2+, S-adenosylmethionine Cofactors
Bipartite & asymmetric Recognition site Type I
Nonspecific>1000 bp from
recognition site Cleavage site
EcoAI, EcoKI
Examples
Unifunctional enzyme
Either with endonuclease/ Structure
methylase activity
Two identical subunits Subunits
Mg2+ Cofactors
Also Known as"Restriction
4-6 bp sequence, often Type II Classification
Endonuclease/Molecular Scissors"
palindromic Recogntiton site
Belong to larger class of enzymes
Same as or close to recognition called “Nucleases”
site Cleavage site Introduction
Recognizes specific base pair
EcoRI, BamHI Example
sequence in DNA called
Bifunctional enzyme “Restriction site”
Structure Cleaves DNA within sequence
Endonuclease & methylase activity
Two different subunits Subunits
ATP, Mg2+ Cofactors
Recognition site Type III RESTRICTION ENZYMES (RE)
5-7 bp Asymmetric sequence
24-26 bp downstream of
recognition site Cleavage site
HinfIII, EcoPI Examples

Both strands of DNA

Examples
Cut at same place in middle of
Blunt ends
recognition site
Produces "Blunt ends/Flush ends"
Both strands of DNA
Not cut at same position
Cleavage staggered, usually by
two or four nucleotides Sticky Ends
Resulting DNA fragments have
short single-stranded overhangs at Cleavage Pattern
each end
Produces “Sticky/Cohesive ends”
Stick back by transient base
pairing

Named according to organism in

which they were discovered
Using system of letters &
numbers
First three letters of the name are
italicized
Abbreviate genus & species Nomenclature
names of the organism
"H": First letter of Genus name
(Haemophilus)
"in": First two letters of the species
name (influenza)
"d" : strain type Example: HindIII
"III" : Third enzyme discovered in
that organism (Roman numerals
used)
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Factors VII, VIII, IX
Blood Proteins
Tissue plasminogen activator
Urokinase
Epidermal growth factor
Nerve growth factor
Follicle stimulating hormone
Human Hormones
Insulin
Recombinant DNA Products
Relaxin Examples
Somatotropin
α-Interferon; β-interferon
Colony stimulating factor
Immune Modulators
Tumor necrosis factor
Lysozyme
Cytomegalovirus
Hepatitis B
Vaccines DNA molecules from different
Measles Joining organisms
Rabies Inserting into host organism

What Is It? To produce new genetic combinations

breaking phosphodiester bonds Degrade DNA molecules Nucleases
science
Recognizes specific base pair
cleaves DNA within sequence sequence in DNA Restriction Enzymes medicine
Enzymes Value to
To synthesize Polymerases agriculture
RECOMBINANT DNA industry
Modify DNA Modifying Enzymes
TECHNOLOGY (RDT)
Join 2 dsDNA molecules Ligases
Isolate DNA from 2 sources
gene inserted to construct DNA molecule capable of How to Construct Recombinant with same Restriction
recombinant DNA molecule replication in host organism
DNA Molecule? Cut DNA Endonuclease
pBR322 DNA ligase covalently links 2 into the molecule of recombinant
Example Plasmids
pUC8 Join DNA strands DNA
pJB8 Example Cosmids
Lambda
Example Bacteriophages
M13 Vectors

Bacterial Artificial Chromosome

(BAC)
Yeast Artificial Chromosome
(YAC)
Recombinant DNA Technology
P1-derived Artificial Example Artificial chromosome
Tools
Chromosome (PAC)
Mammalian Artificial
Chromosome (MAC)

Harbors foreign molecules

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(rDNA)
Non-pathogenic, easy to
cultivate

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Host Organism
Bacterial cell (most commonly
E.coli)
Plant cell
Animal cell
Example
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Introduction of any DNA
molecule into any living cell Starts 2nd March 2023
Natural
Microinjection
Transformation Methods
Biolistics Physical
Electroporation
Calcium phosphate Artificial
Chemical
Liposomes
A. tumefaciens
Biological
A. rhizogenes
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Cell frequencies bind to

fluorescent antibodies &
Quantifies scattering light in
characteristic ways
at number of different
level of emitted fluorescence wavelengths
ANALYTICAL TECHNIQUE
Record amount of forward- & side-
scattered light for each cell
When adapted to sort cell on basis of fluorescence & light referred to as Fluorescence
subpopulations scattering Activated Cell Sorter (FACS)

Transport particles in fluid

Fluidic System stream to laser beam for interrogation
Lasers
Excitation optics
Lenses
allow light within certain range
FLOW CYTOMETRY of wavelengths eg: 520-550 nm to pass
Routinely described by pair of
numbers
Band pass (BP) filters
First: denotes central
wavelength of BP filter

Example: 530/30 Second :shows range of on either side of that central that will be allowed to pass
wavelengths wavelength through filter

allow passage of any light of

wavelengths longer than filter specification
Optical System
reflect (or stop) light of shorter
Filters
wavelengths
Long pass (LP) filters
allow any wavelengths longer
than 640 nm to pass through
Example: 640 LP filter refl ect light of wavelengths
COMPONENTS OF FLOW shorter than 640 nm
CYTOMETER letting wavelengths lower than
filter specification pass
Short pass (SP) filters operate in converse manner
reflecting higher wavelengths

reflect light of specific

Collection optics wavelengths
allowing all other light to pass
Dichromic mirrors
can also be referred to as SP/ depending on their
LP characteristics
Forward scattered (FSC) light
Capture & Detect
Not particularly sensitive

Photodiodes reflect light of specific

wavelengths

Dichroic mirrors allowing all other light to pass

Detectors depending on their

can also be referred to as SP/LP characteristics
Side scattered (FSC) light
Capture & Detect
Photomultiplier Tubes (PMTs) Fluorescence signals
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from detectors into digital
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Immunophenotyping
Measuring intracellular cytokine
production
Cellular proliferation Analysis
Apoptosis Analysis
Cell cycle Analysis
Cell Sorting
Single Parameters Analysis
Fluoroscein; Alexa Fluor 488;
Small Organic Molecules Example
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Texas Red; Pacific Blue; Cy5
Cyanobacteria, Dinoflagellates, &
large protein molecules derived from Algae
Phycoerythrin (PE);
Phycobiliproteins
Allophycocyanin (APC); Peridinin
Example Chlorophyll Protein (PerCP)
To measure cell proliferation
Proliferation Dyes
Example Bromodeoxyuridine (BrdU)
can be “tuned” to absorb & emit at specific wavelengths made to
light absorb light
very stable
have similar quantum efficiency
FLUOROCHROMES USED to phycobiliproteins
Polymer Dyes
greatly increased photostability
Brilliant Violet (BV); Brilliant
Ultraviolet (BUV); Brilliant Blue
Example (BB)
reporter systems for gene
frequently used as expression
Fluorescent Proteins
Example GFP
Bind DNA, RNA or both
Quantitate DNA for cell cycle analysis
Discriminate chromosomes for sorting
APPLICATIONS
For sorting stem cells
Nucleic Acid Dyes Bacteria
Cell viability
Propidium Iodide; DAPI;
Hoescht 33342, Chromomycin
Example A3

Identify any fluorescent marker

expression within population
Fast to read & easy to
understand
Dual Parameters Analysis
Combinations of dyes analysed
simultaneously
Density Plots
Each distinct event represented
at single dot
More complex
8-12 fluorescence & light-
scattering parameters
routinely used in clinical &
research laboratories
1) Cells (suspension) introduced
Histogram
into sample injection port
2) Focused within stream of
sheath fluid
DATA ANALYSIS FLOW CYTOMETRY
3) Pass one by one in front of
laser beam
4) Forward scattered light detected by photodiode measure cell size
HOW IT WORKS
detected by photomultiplier after passage through a series Dichroic mirrors
tubes of
Light filters
at 90 degree to path of laser
placed beam
5) Side-scattered light & extent of intracellular
CAPABLE OF DETECTING emitted fluorescence of various indicate complexity of scattering cell
wavelengths Side scatter detector
more intracellular membranous
structures present in scattering cell
more side-angle light scatter Example: Endoplasmic reticulum
(ER) & Mitochondria

6) All information obtained from Can be expressed in number of

individual cells format integrated by software

of fluorochrome-labeled cells
withflow cytometer

SEPARATION

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Best Resolution 0.5 nm
Radiation Source Electron Beam
Medium of Travel High Vacuum
Introduction Type of Lens Electromagnet
Source of Contrast Scattering of electrons
Focusing Mechanism Adjust current to magnetic lens
Method Of Changing Magnification Adjust current to magnetic lens
Specimen Mount Metal grid (usually copper)

Examine surfaces of microorganisms

Resolution 7 nm/less
By scanning sample with focused beam
of electrons
SEM Produce Image
Image produced from electrons emitted by object’s surface
TEM has much higher resolution than Scanning Electron Microscope (SEM) (i) Electron beam strikes particular
SEM Resolution area

In SEM electrons emitted by object's (ii) Surface atoms discharge tiny

surface form image shower of electrons called secondary electrons

Image (iii) Trapped by special detector

In TEM transmitted electrons form
image How it Works (iv) Secondary electrons entering
SEM vs TEM detector strike scintillator
ELECTRON MICROSCOPY
SEM provides 3-D picture
Type of Image (v)Photomultiplier converts to
TEM provides 2-D picture electrical current & amplifies signal sent to cathode-ray tube

TEM requires extensive sample (vi) Produces imge like televisison

preparation Sample Preparation picture can be viewed/photographed
Electron beam transmitted through
specimen forms image
(i) Electron beam generated by tungsten filament in electron gun
Transmission Electron Microscope (ii) Focused on specimen by condenser
(TEM)
(iii) Specimen scatters electrons
passing through it

Denser region in specimen scatters appears darker in image

How It works
more electrons as fewer electrons strike area of
(iv) Image Produced screen

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(v) Image captured on photographic
film as permanent record
Types Of Electron Microscope

Monthly Fees Structure is also Available; chat

Allow scientists to view atoms on solid
surface

with us to know more Principle of Operation Quantum mechanical phenomenon

(i) Needle like probe
known as Tunneling

until its electron cloud touches surface

(ii) Lowered toward specimen surface atoms

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Scanning Tunneling Microscope
(iii) Small voltage applied between tip &
specimen

(iv) Electrons flow thorugh narrow

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channel in electron clouds called tunneling current sensitive to distance

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tunnel/project out from surface
(v) Electrons surrounding surface atom boundary

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(vi) Arrangement of atoms on specimen moving probe tip back & forth over keeping at constant height
surface determined surface
to maintain steady tunneling current

Video Pen Drive (vii) Computer record & analyze tip

motion to create 3D image of surface atoms
(viii) Surface map displayed on
computer screen/plotted on paper
Measures small force between surface
& tip

Used study surfaces

Atomic Force Microscope
(i) Moves sharp probe over specimen
(ii) Keep distance between probe tip &
How It Works? surface constant
(iii) Tip vertical motion followed by
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Not safe
Disadvantage
More costly then ELISA

for presence of Hepatitis B
virus
to fixed quantity of
[125I]HBsAg & specific antibody
using linear part of curve
Hormones
Serum proteins Measure S.A. Berson & Rosalyn Yalow
Drugs (1960)
Applications
Developed by to determine levels of insulin/
Screening of Donor blood anti-insulin complexes in diabetic patients
Highly sensitive & quantitative
adding increasing concentrations procedure
of unlabeled HBsAg Obtained by
Utilizes radioactively labeled
Percentage of labeled antigen Introduction antigen/antibody
bound versus Concentration of
unlabeled antigen that involves competitive
Use to measure antigen/ binding of radiolabeled antigen/
HBsAg concentration in unknown Standard Curve antibody concentration antibody
serumsamples determined
Detects as low as few picograms
of analyte

Involves combination of 3
From plot of principles

1) Strong immune reaction Antigen, antibody binding

2. Competitive Binding/
Principle of Radioimmunoassay Competitive Displacement
RADIOIMMUNOASSAY (RIA)
Reaction It gives specificity

3) Measurement of radio
emission It gives sensitivity
surface antigen on hepatitis B with constant amount of
virions antibody specific for HBsAg 1) Microtiter wells coated Competitive Binding/Competitive Two antigens that can bind to binds extensively with limited
After incubation, supernatant 2) Serum sample & [125I]HBsAg Displacement Reaction same antibody antigen with more concentration antibody displacing others
removed added
3) Radioactivity of antigen- Gamma-emitting isotope 125I
Isotopes Used
antibody complexes measured Detection of Hepatitis B virus in Beta-emitting isotopes Tritium (3H)
will be less than in controls with blood samples Procedure
uninfected serum amount of label bound 4) If sample infected Serum or
Test Sample Complex mixture
Other body fluids that contains unlabeled antigen

after beads havebeen Amount of radiolabeled antigen A) Antibody covalently

centrifuged & washed bound to beads can be measured crosslinked to Sepharose beads

determined in radiation counter
B) Antibody immobilized on
Amount of free labeled antigen polystyrene/ polyvinylchloride
in supernatant wells
Solid-Phase RIAs
Amount of bound antigen
determined

Requires only small amounts of C) Antibody immobilized on walls

sample of microtiter wells

of particular antigen in large Well suited for determining Can be conducted in small 96-
numbers of samples concentration well microtiter plates

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