Molecular techniques are major tools for the analysis of microorganisms. Molecular methods varies with respect to discriminatory power, reproducibility, ease of use, and ease of interpretation.

1. VEENSA
VE
MOLECULAR IDENTIFICATION OF BACTERIAL
PATHOGENS
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2. • Molecular techniques are major tools for the
analysis of microorganisms.
• Molecular methods varies with respect to
discriminatory power, reproducibility, ease of
use, and ease of interpretation.
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3. ELECTROPHORESIS
•Electrophoresis is a method where by charged
molecules in solution, chiefly proteins and
nucleic acids, migrate in response to an
electrical field.
•Their rate of migration through the electrical
field, depends on the strength of the field, on
the net charge, size, and shape of the
molecules, and also on the ionic strength,
viscosity, and temperature of the medium in
which the molecules are moving.
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4. •As an analytical tool, electrophoresis is simple,
rapid and highly sensitive.
•It can be used analytically to study the
properties of a single charged species or
mixtures of molecules. It can also be used
preparatively as a separating technique
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5. •Electrophoresis is usually done with gels
formed in tubes, slabs, or on a flat bed.
•In many electrophoresis units, the gel is
mounted between two buffer chambers
containing separate electrodes, so that the
only electrical connection between the two
chambers is through the gel.
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6. • In most electrophoresis
units, the gel is
mounted between two
buffer chambers
containing separate
electrodes so that the
only electrical
connection between
the two chambers is
through the gel.
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7. AGAROSE GEL
• Agarose is a highly purified uncharged
polysaccharide derived from agar
• Agarose dissolves when added to boiling liquid. It
remains in a liquid state until the temperature is
lowered to about 40° C at which point it gels
• The pore size may be predetermined by adjusting
the concentration of agarose in the gel
• Agarose gels are fragile, however. They are
actually hydrocolloids, and they are held together
by the formation of weak hydrogen and
hydrophobic bonds
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8. Polyacrylamide gels
• Polyacrylamide gels are tougher than agarose
gels
• Acrylamide monomers polymerize into long
chains that are covalently linked by a
crosslinker
• Polyacrylamide is chemically complex, as is the
production and use of the gel
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10. PCR
• PCR targets and amplifies a specific region of
a DNA strand.
• It is an in-vitro technique to generate large
quantities of a specified DNA.
• PCR is ‘photocopier
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12. THERMOCYCLER
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13. REQUIREMENTS
• DNA Template
• Primers
• Taq polymerase
• Deoxynucleoside triphosphates(dNTPs)
• Buffer solution
• Divalent cations(eg.Mg2+)
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14. Inverse PCR
• Amplification of DNA of
unknown sequence
carried out from known
sequence.
• identification of
sequences flanking
transposable elements
• identification of
genomic inserts
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15. REVERSE TRANSCRIPTION PCR (RT-
PCR)
• For amplifying DNA
from RNA.
• Reverse transcriptase
reverse transcribes
RNA into cDNA, which
is then amplified by
PCR.
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16. Quantitative real time PCR(Q-RT PCR)
• It quantitatively measures
starting amounts of DNA,
cDNA or RNA.
• Q-PCR is commonly used
to determine whether a
DNA sequence is present in
a sample and the number of
its copies in the sample.
• QRT-PCR methods use
fluorescent dyes, such as
Sybr Green, EvaGen to
measure the amount of
amplified product in real
time.
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18. DETECTION
• Detection is based on fluorescence
technology
• the marker added to the sample and the
signal is amplified with the amplification of
copy number of sample DNA.
• emitted signal is detected by an detector
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19. • There are many different markers used as the
marker of Real Time PCR.
• There are mainly two types of marker are used
for this purpose.
1.Taqman probe.
2.SYBR Green
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20. MULTIPLEX PCR
• Multiplex PCR is a widespread molecular
biology technique for amplification of multiple
targets in a single PCR experiment.
• In a multiplexing assay, more than one target
sequence can be amplified by using multiple
primer pairs in a reaction mixture.
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22. NESTED PCR
• Nested PCR is used to increase the sensitivity of
detecting pathogens, such as Neorickettsia risticii, that
may not be detectable after 30 to 40 cycles of regular
PCR.
• Unlike regular PCR, nested PCR uses two pairs of
primers and two sequential series of PCR amplification.
• The first amplicon is created using the first (outer) pair
of primers.
• This initial amplicon is then used as a template for the
second PCR, which uses the second (inner) primer pair
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24. REP PCR
• REP = short sequences that are occur in
multiple locations throughout the bacterial
genome
• REP-PCR assays variation in sequence at
multiple sites throughout the genome
• Patterns differentiate bacteria at
subspecies level
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• Is used for Genomic Fingerprinting of plant-
associated bacteria and computer-assisted
plant analyses. The genomic fingerprinting
method employed is based on the use of DNA
primers corresponding to naturally occurring
interspersed repetitive elements in bacteria
such as REP,ERIC and BOX elements.

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27. ERIC PCR
• ERIC sequences are also of interest because
they have been used as the basis of a
technique for fingerprinting bacterial genomes
(Versalovic, Koeuth, and Lupski
1991). Polymerase chain reaction(PCR)
primers were designed to amplify between
copies of the ERIC sequence at nearby
locations in the bacterial genome.
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30. RAPD
• RAPD (pronounced as "rapid") stands for
'Random Amplification of Polymorphic DNA'.
• ]It is a type of PCR, but the segments of DNA
that are amplified are random.
• No knowledge of the DNA sequence of the
targeted genome is required, as the primers
will bind somewhere in the sequence, but it is
not certain exactly where.
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32. RFLP
• The term Restriction Fragment Length
Polymorphism , or RFLP refers to a difference
between two or more samples of homologous
DNA molecules arising from differing locations
of restriction sites, and to a related laboratory
technique by which these segments can be
distinguish .
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34. • Terminal restriction fragment length
polymorphism (TRFLP or sometimes T-
RFLP)
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35. • T-RFLP analysis is a technique used to study complex
microbial communities based on variation in the 16S
rRNA gene .
• T-RFLP analysis can be used to examine microbial
community structure and community dynamics in
response to changes in different environmental
parameters or to study bacterial populations in
natural habitats.
• It has been applied to the study of complex
microbial communities in diverse environments such
as soil , marine and activated sludge systems
• as well as in a study to characterize oral bacterial
flora in saliva in healthy subjects versus patients with
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37. Amplified Fragment Length
Polymorphisms (AFLPs)
PRINCIPLE OF AFLP
• The AFLP technique is based on the principle
of selectively amplifying a subset of restriction
fragments from a complex mixture of DNA
fragments obtained after digestion of genomic
DNA with restriction endonucleases.
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38. STEPS
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39. Multi locus sequence typing (MLST)
• This is a technique in molecular biology for the typing
of multiple loci. The procedure characterizes isolates of
microbial species using the DNA sequences of internal
fragments of multiple housekeeping genes.
• Approximately 450-500 bp internal fragments of each
gene are used, as these can be accurately sequenced
on both strands using an automated DNA sequencer.
• For each housekeeping gene, the different sequences
present within a bacterial species are assigned as
distinct alleles and, for each isolate, the alleles at each
of the loci define the allelic profile or sequence type
(ST).
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41. RIBOTYPING
• Ribotyping involves the digestion of bacterial genomic
DNA with specific restriction enzymes. Each restriction
enzyme cuts DNA at a specific nucleotide sequence,
resulting in fragments of different lengths.
• Those fragments are then run on a Gel electrophoresis,
where they are separated according to size: the
application of electrical field to the gel in which they
are suspended causes the movement of DNA
fragments (all negatively charged due to the presence
of phosphate groups) through a matrix towards the
positively charged end of the field. Small fragments
move more easily and rapidly through the matrix,
reaching a bigger distance from the starting position
than larger fragments.
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42. • Following the separation in the gel matrix, the
DNA fragments are moved onto nylon
membranes and hybridized with a labelled 16S
or 23S rRNA probe. This way only the
fragments coding for such rRNA are visualised
and can be analyzed.The pattern is then
digitized and used to identify the origin of the
DNA by a comparison with reference
organisms in a computer database.
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