MODERN SYSTEMS OF BACTERIAL

TAXONOMY
DNA ANALYSIS
NUCLEIC ACID HYBRIDIZATION
Traditional vs Modern
Binomial taxonomy DNA sequences
Modern systems
The routine laboratory identification are the
microscopic observation/ colonies
morphology/ biochemical test
Latest and the most modern era of
identification are molecular biological test
Most of the methods relates -genetic analysis
Also other methods


Serology
Studies of serum and immune responses
m/o is antigenic- produce response as
antibodies
The reagent used in test kit is antiserum
Present of m/o usually causing the clotting
effects towards antiserum (+ve result)
Oppositely, the absent of m/o will be diluted
effects (-ve result)
Types of serology test
Slide agglutination test
ELISA (Enzyme-linked Immunosorbent
Agglutination)
Phage typing
Phage typing is a method used for detecting
single strains of bacteria.
It is used to trace the source of outbreaks of
infections.
[1]

The viruses that infect bacteria are called
bacteriophages ("phages" for short) and
some of these can only infect a single strain
of bacteria.


These phages - used to identify different strains
of bacteria within a single species.
A culture of the strain is grown in the agar and
dried
A grid is drawn on the base of the petri dish to
mark out different regions.
Inoculation of each square of the grid is done by
a different phage.
The phage drops are allowed to dry and are
incubated:
The susceptible phage regions - circular clearing
where the bacteria have been lysed- used in
differentiation.

A culture of bacteria infected by
bacteriophages, the "holes" are areas
where the bacteria have been killed by the
virus. The culture is 10cm in diameter
Fatty acid analysis
Also known as FAME (fatty acid methyl ester)
Fatty acid methyl esters (FAME) are a type
of fatty acid ester that can be produced by an
alkali-catalyzed reaction between fats or
fatty acids and methanol.
The molecules in biodiesel are primarily
FAMEs, usually obtained from vegetable oils
by transesterification.
Every microorganism has its specific FAME
profile (microbial fingerprinting)
A tool for microbial source tracking (MST).
To perform this analysis,
a bacterial culture is taken
fatty acids extracted
form methyl esters (volatile)
gas chromatagraph (observe peak/pattern)

This is widely used in characterizing new
species of bacteria, and is useful for
identifying pathogenic strains.
Flow cytometry
Flow cytometry is a laser based, biophysical
technology employed in cell counting, cell
sorting, biomarker detection and protein
engineering, by suspending cells in a stream of
fluid and passing them by an electronic detection
apparatus.
To identify bacteria in sample without culturing
the bacteria
Cell and m/o go through a small portion
Pass thru a laser and show scattering graph
determining size, shape, density and surface
Also consist of fluorescent detection








schematic diagram of a flow cytometer, showing
focusing of the fluid sheath, laser, optics (in
simplified form, omitting focusing),
photomultiplier tubes (PMTs), analogue-to-digital
converter,
DNA base composition
Show the relatedness via % of (Guanine G and
cytosine C)
G complement with C, A complement with T
Pair s of GC resulting pairs of AT; (GC + AT =
100%)
Eg. If there are 2 m/o, might be 1
st
have
40%GC but the other have 80%GC

DNA Fingerprinting

Determine the specific sequences of
bacteria(pathogenic).
Apply DNA enzyme restriction.
It can cut molecule of DNA everywhere,
specific sequence.
By comparing the specific seq. from samples
and standard (known bacteria), one can
know/expect the unknown.

Eg- determine the source of hospital-
acquired infections.
DNA fingerprinting is used.
The hospital personnel sample is compared
to patient sample.
Relate to hospital personnel or patients from
the same ward
Short-time period required, instead of routine
process
Should have the control sequences
Similar sequences shows >% of relationship


Polymerase Chain Reaction
m/o cannot be cultured through conventional
methods
This method can be used to increase the
amount of microbial DNA to levels that can
be tested by gel electrophoresis
Eg. From the of dinosaur/ancient sample
If there are primer of particular m/o in the
sample, there will be amplified DNA indicates
that m/o, obviously shows in the gel
Southern Blotting
DNA chips
Ribotyping and Ribosomal RNA sequencing
Flourescent In Situ Hybridization (FISH)
Nucleic acid hybridization
Introduction
Each complimentary DNA containing
complementary bases
When heat, the hydrogen bonds separated
When cool, it reunite almost back to normal
Basic purpose is to identify unknown m/o
Can hybridize any single-stranded nucleic
acid chain; DNA-DNA, RNA RNA, DNA-RNA
Nucleic acid hybridization
Nucleic acid hybridization
What is blotting?
Blots are techniques for transferring DNA ,
RNA and proteins onto a carrier so they can
be separated, and often follows the use of a
gel electrophoresis.
TYPES OF BLOTTING TECHNIQUES

Blotting technique

Southern Blot

It is used to detect DNA.

Northern Blot

It is used to detect RNA.
Western blot

It is used to detect protein.
SOUTHERN BLOTTING
Professor Sir Edwin Southern,
Professor of Biochemistry and
Fellow of Trinity developed this
method in 1975.
Southern won the Lasker
Award for Clinical Medical
Research prize for the method
of finding specific DNA
sequences he developed this
procedure at Edinburgh University
more than 30 years ago. The
technique is known as DNA
transfer or 'Southern blotting'

Professor Sir Edwin Southern
Cont.
This method Involves separation, transfer and
hybridization.
It is a method routinely used in molecular biology for
detection of a specific DNA sequence in DNA samples. The
DNA detected can be a single gene, or it can be part of a
larger piece of DNA such as a viral genome.

Cont.
Southern blotting combines agarose gel electrophoresis
for size separation of DNA with methods to transfer the
size separated DNA to a filter membrane for probe
hybridization.
The key to this method is Hybridization.
Hybridization - Process of forming a double-stranded
DNA molecule between a single-stranded DNA probe
and a single-stranded target patient DNA.

PRINCIPLE
1. The mixture of molecules is separated.
2. The molecules are immobilized on a matrix.
3. The probe is added to the matrix to bind to the
molecules.
4. Any unbound probes are then removed.
5. The place where the probe is connected
corresponds to the location of the immobilized target
molecule.


APPARATUS





Whatman 3MM paper
nitrocellulosemembrane
Capillary plotting apparatus
Steps in southern blotting
1. Digest the DNA with an
appropriate restriction
enzyme.

2.The complex mixture of
fragments is subjected to gel
electrophoresis to separate
the fragments according to
size.

Cont.
3.The restriction fragments
present in the gel are
denatured with alkali and
transferred onto
4. a nitrocellulose filter or nylon
membrane by blotting.

This procedure preserves the
distribution of the fragments
in the gel, creating a replica
of the gel on the filter.

Cont.
5.The filter is incubated under
hybridization conditions
with a specific radiolabeled
DNA probe.

The probe hybridizes to the
complementary DNA
restriction fragment.

Cont.
6.Excess probe is washed away and the
probe bound to the filter is detected
by autoradiography, which reveals the
DNA fragment to which the probe
hybridized.

APPLICATIONS
Southern blots are used in gene discovery , mapping,
evolution and development studies, diagnostics and
forensics (It is used for DNA fingerprinting, preparation of RFLP
maps)
identification of the transferred genes in transgenic individuals, etc.
Application
Salmonella DNA fragment is cloned in E.coli
Cloned DNA fragment is extracted and
labeled with fluorescence dye
Then the procedure for identification
(diagram) are repeated
DNA probes hybridize with salmonella DNA
from sample. Excess probe is washed

APPLICATIONS
Southern blots allow investigators to determine the
molecular weight of a restriction fragment and to
measure relative amounts in different samples.
Southern blot is used to detect the presence of a
particular bit of DNA in a sample
analyze the genetic patterns which appear in a person's
DNA.

Northern Blotting
Northern blotting is a technique for detection of
specific RNA sequences. Northern blotting was
developed by James Alwine and George Stark at
Stanford University (1979) and was named such by
analogy to Southern blotting
Steps involved in Northern
blotting
1. RNA is isolated from several
biological samples (e.g.
various tissues, various
developmental stages of same
tissue etc.)
* RNA is more susceptible to
degradation than DNA.
Cont
2. Samples are loaded on gel and the
RNA samples are separated according
to their size on an agarose gel .

The resulting gel following after the
electrophoresis run.

Cont
3. The gel is then blotted on a
nylon membrane or a
nitrocellulose filter paper by
creating the sandwich
arrangement.

Cont
4. The membrane is placed in a dish
containing hybridization buffer with a
labeled probe.
Thus, it will hybridize to the RNA on
the blot that corresponds to the sequence
of interest.

5. The membrane is washed to remove
unbound probe.

Cont
6. The labeled probe is detected via
autoradiography or via a
chemiluminescence reaction (if a
chemically labeled probe is used). In
both cases this results in the
formation of a dark band on an X-ray
film.
Now the expression patterns of the
sequence of interest in the different
samples can be compared.

APPLICATIONS
A standard for the study of gene expression at the level of mRNA
(messenger RNA transcripts)
Detection of mRNA transcript size
Study RNA degradation
Study RNA splicing
Study RNA half-life
Often used to confirm and check transgenic / knockout mice
(animals)

Western blotting
Western blotting (1981) is an Immunoblotting technique which rely on
the specificity of binding between a protein of interest and a probe
(antibody raised against that particular protein)
to allow detection of the protein of interest in a mixture of many other
similar molecules.

The SDS PAGE technique is a prerequisite for Western blotting .

Steps in western blotting
1. A protein sample is subjected to
electrophoresis on an SDS-
polyacrylamide gel.

2. Electroblotting transfers the
separated proteins from the gel
to the surface of a nitrocellulose
membrane.

Cont
3. The blot is incubated with a generic protein (such as milk
proteins or BSA) which binds to any remaining sticky
places on the nitrocellulose.

4. An antibody that is specific for the protein of interest (the
primary antibody - Ab1) is added to the nitrocellulose
sheet and reacts with the antigen. Only the band
containing the protein of interest binds the antibody,
forming a layer of antibody molecules .

Cont
5. After washing for removal of non-specifically
bound Ab1, second antibody (Ab2)is added,
which specifically recognizes the Fc domain of
the primary antibody and binds it. Ab2 is
radioactively labeled, or is covalently linked to
a reporter enzyme, which allows to visualize
the protein-Ab1-Ab2 complex.

An example
Application
1.The confirmatory HIV test
2.Western blot is also used as the definitive test
for Bovine spongiform encephalopathy (BSE(
3.Some forms of Lyme disease testing employ Western
blotting .

DNA chips
Possible to quickly detect a pathogen in a
host by identifying a gene that is unique to
that pathogen
The chips composed of DNA probe
Unknown DNA label by fluorescent dye and
placed in a chips
Hybridization between DNA probe and DNA
detect by fluorescent
DNA chip technology
Fluorescent In Situ
Hybridization
Fluorescent dye-labeled RNA or DNA probe
are used to specifically m/o in place or in situ
Cell will be treated then the probe will enter
the cell easily and react to the target
ribosome in the cell (in situ)
To determine the identity, abundance and
relative activity of m/o in an environment
Also to detect bacteria that have not yet been
cultured (tiny quantity)
FISH
Ribotyping and Ribosomal RNA
Sequencing
To determine the phylogenic relationships
among organisms
Use of small sub unit rRNAs (part of complex
ribosome DNA)
16s rRNA for Bacteria, 18s rRNA for fungi.
3 advantages:
All cells contain ribosomes
RNA genes cannot tolerate mutation
Highly conserved among species
Cell do not have to be cultured in laboratory

The steps
Genomic DNA Isolation
Gel electrophoresis
Polymerase chain reaction (PCR) -
PCR product purification -
Sequencing of the 16S rRNA Gene-
Obtaining full sequence of bacteria

Homology Search- (BLASTn) National Centre of
Biotechnology Information (NCBI) website
(http://www.ncbi.nlm.nih.gov).

Construction of phylogenetic tree-
(special)programming, eg DotPLot

Ribosomal sequencing
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A B C M
Agarose gel electrophoresis analysis of the
16S rDNA gene fragment after purification
amplified using universal primers