Presentation

on

Molecular Methods
of
Characterization of Microorganisms
Characterization is important for identification &
classification of MO.

Important in the field of environmental, industrial, medical

and agricultural microbiology & microbial ecology.

To investigate disease causing microorganism.

Selective extraction of individual microorganisms from

native microbial communities,

To detect, isolate & characterize MO capable of degrading

the harmful substances in environment (viz. pesticides)
 Methods for Characterization of
Microorganisms
• Morphological methods
• Bio-Chemical methods

• Molecular methods
 Molecular Methods

• Polymer Chain Reaction (PCR )method

• Denaturing Gradient Gel Electrophoresis (DGGE)

• Pulsed-Field Gel Electrophoresis (PFGE)

• Fluorescence in-situ hybridization(FISH)

 Target Microorganisms for
Molecular-Based Testing
• Those that are difficult or time-consuming to isolate
– e.g., Mycobacteria
• Hazardous organisms
– e.g., Histoplasma, Coccidiodes
• Those without reliable testing methods
– e.g., HIV, HCV
• High-volume tests
– e.g., S. pyogenes, N. gonorrhoeae, C. trachomatis
 Molecular Methods
• Molecular methods use one of several forms of
complementarity to identify the macromolecules of
interest among a large number of other molecules.
• Complementarity is the sequence-specific or shape-
specific molecular recognition that occurs when two
molecules bind together.
– two strands of a DNA double-helix bind because they have
complimentary sequences
– an antibody binds to a region of a protein molecule because
they have complimentary shapes.
 Hybrid Molecular Complexes
(Hybrids)
• DNA-DNA: A single-stranded DNA (ssDNA) probe
molecule can form a double-stranded, base-paired
hybrid with a ssDNA target if the probe sequence is
the reverse complement of the target sequence.
• DNA-RNA: A ssDNA probe molecule can form a
double-stranded, base-paired hybrid with an RNA
(RNA is usually a single-strand) target if the probe
sequence is the reverse complement of the target
sequence.
• Protein-Protein: An antibody probe molecule can
form a complex with a target protein molecule if
the antibody's antigen-binding site can bind to an
epitope (small antigenic region) on the target
protein. In this case, the hybrid is called an
'antigen-antibody complex' or 'complex' for short.
 Important features of
hybridization
1) Hybridization reactions are specific - the probes will only bind
to targets with complimentary sequence (or, in the case of
antibodies, sites with the correct 3-D shape).
2) Hybridization reactions will occur in the presence of large
quantities of molecules similar but not identical to the target.
That is, a probe can find one molecule of target in a mixture of
zillions of related but non-complementary molecules.

These properties allow you to use hybridization to perform a

molecular search for one DNA molecule, or one RNA molecule,
or one protein molecule in a complex mixture containing many
similar molecules.
 Importance of hybridization
techniques
• Cell contains tens of thousands of genes, thousands of different
mRNA species, and thousands of different proteins.
• When the cell is broken open to extract DNA, RNA, or protein,
the result is a complex mixture of all the cell's DNA, RNA, or
protein.
• It is impossible to study a specific gene, RNA, or protein in such
a mixture with techniques that cannot discriminate on the basis
of sequence or shape.
• Hybridization techniques allow you to pick out the molecule of
interest from the complex mixture of cellular components and
study it on its own.
 Summary of blotting techniques
Southern Blot: For analysis of DNA- Generally used to
determine size, presence and copy number.
(Probe = DNA fragment).

Northern Blot: For Analysis of RNA – Can determine

presence, size of transcript and steady-state level.
(Probe = DNA or RNA).

Western Blot: For Analysis of protein – Can determine

presence, size of protein and steady-state level.
(probe = antibody).
 General steps
• Gel electrophoresis
• Transfer to Solid Support
• Blocking
• Preparing the Probe
• Hybridization
• Washing
• Detection of Probe-Target Hybrids
 Specimen Collection
• Preserve viability/nucleic acid integrity of target
microorganisms
• Avoid contamination
• Appropriate time and site of collection (blood, urine,
other)
• Use proper equipment (coagulant, wood, or plastic
swab shafts)
• Commercial collection kits are available
• The Clinical and Laboratory Standards Institute
(CLSI) has guidelines for proper specimen handling
 Sample Preparation
• Consider the specimen type (stool, plasma, CSF)

• More rigorous lysis procedures are required to penetrate

cell walls

• Consider the number of organisms in the sample

• Inactivate inhibitors (acidic polysaccharides in sputum

or polymerase inhibitors in CSF)

• Inactivate RNases
 Denaturing Gradient Gel
Electrophoresis (DGGE)
• Direct visualization of bacterial diversity

• Identification of community members

• By sequence analysis of excised bands

• By hybridization experiments using taxon specific probes

• Low cost and consume less time

Overview of DGGE Analysis
 Applications of DGGE

Study community complexity

Monitor population shifts
Analyze enrichment cultures and the isolation of bacteria
Detect sequence heterogeneities of RNA genes in single
genomes
Compare DNA extraction methods
Screen clone libraries
Determine PCR and cloning biases
 Northern blotting
• Detects the presence (and gives
us the size of) a specific mRNA in
a total RNA extract.
• Can determine if a gene is
transcribed or not and determine
where and when it is transcribed.
• Total RNA resolved on a
denaturing agarose
gel,transferred to a membrane
and immobilized for subsequent
hybridization.
 Northern blot – RNA on gel
Total RNA from different tissues

agarose gel
stained with
ethidium bromide rRNA
rRNA

autoradiograph of
northern blot after
hybridization with
radioactive probe

Northern Blots can tell you in what tissue a gene is expressed, and how much
of the gene is expressed. Also useful for detection of splice variants.
Northern Blot probed with b-globin

Note: Make northern just like a Southern,

except run out RNA samples rather than DNA
• Measures the steady-state level of mRNA
transcript from a given gene

Smear of RNA transcripts

Undifferentiated + differentiation factor

cells
• Same Northern blot may be stripped (boiled in 100mM NaOH)
and re-probed with different probes several times.
• It is often prudent to use a probe specific to a “housekeeper” or
“constitutively” expressed gene such as GAPDH
(Glyceraldehyde-3-phosphate dehydrogenase) or β-Actin to
“normalize” the blot.
• Normalising ensures that the amount of RNA in lanes are
comparable.
 Western Blotting
• Based on protein-protein interaction
• Detects the presence of (and gives us the size of) a
specific protein in a crude protein extract.
• Can be used to determine if a gene is expressed
(transcribed and translated) or not.
• Involves SDS-PAGE, electrophoretic transfer to
membrane, probing with antibodies and detection
 SDS-PAGE
• SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel
electrophoresis, is a technique widely used in
biochemistry, forensics, genetics and molecular biology:
• To separate proteins according to their electrophoretic
mobility (a function of length of polypeptide chain or
molecular weight).
• To separate proteins according to their size, and no other
physical feature.
 SDS-PAGE
Separating proteins by size
• SDS (anionic detergent) breaks up
hydrophobic areas and coats
proteins with negative charges
thus overwhelming positive
charges in the protein.
• The detergent binds to
hydrophobic regions in a constant
ratio of about 1.4 g of SDS per
gram of protein.
• All proteins will be solubilised by
the detergent and will be covered
with many negative charges.
 ..PAGE
• Since all proteins have a large
negative charge they will all
migrate towards the positive
pole when placed in an electric
field.
• Small molecules move through
the polyacrylamide network
faster than big molecules. • The actual bands are equal in
size, but the proteins within
each band may be different
SDS-PAGE
Western blotting
• proteins separated
according to size by
SDS-PAGE
• transferred from the gel
to a filter
• filter (membrane)
incubated with an
antibody against a
protein of interest
• antibody bound to the
filter can be detected
by reaction with various
reagents
 Applications of Molecular Methods
• Molecular-based methods offer sensitive and direct detection of
microorganisms.
• DNA fingerprinting
• DNA typing
• Molecular markers
• Several molecular methods are used to type bacterial strains in
epidemiological investigations.
• Rapid or high-throughput identification of microorganisms
• Detection and analysis of resistance genes
• Genotyping
• Classification
• Discovery of new microorganisms
THANK YOU