MOLECULAR BIOLOGICAL TECHNIQUES

SHALINI MARIA
III YEAR PG
 THE STUDY OF GENE
STRUCTURE AND
FUNCTION AT
THE MOLECULAR

MOLECULA LEVEL.

R BIOLOGY MOLECULAR BIOLOGY

GREW OUT OF THE
DISCIPLINES OF
GENETICS AND
BIOCHEMISTRY
 INTRODUCTION
IN 1865, GREGOR MENDEL
PUBLISHED HIS FINDINGS ON THE
INHERITANCE OF SEVEN DIFFERENT
TRAITS IN THE GARDEN PEA.
EACH PARENT CONTRIBUTES
PARTICLES, OR GENETIC UNITS, TO
THE OFFSPRING.
WE NOW CALL THESE PARTICLES -
GENES.
THE WORD PHENOTYPE, COMES FROM THE SAME
GREEK ROOT AS PHENOMENON, MEANING
APPEARANCE.
THUS, A TALL PEA PLANT EXHIBITS THE TALL PHENOTYPE,
OR APPEARANCE.

PHENOTYPE - THE WHOLE SET OF OBSERVABLE

CHARACTERISTICS OF AN ORGANISM.
 CHROMOSOMES - ORGANS OF HEREDITY
 ATTEMPTS WERE MADE BY EARLY MOLECULAR
GENETICISTS TO IDENTIFY THE PHYSICAL AND CHEMICAL
NATURE OF GENES.

 MINUTE STRUCTURES - PHYSICAL IDENTITY - IMPOSSIBLE.

 EXTENSIVE CHEMICAL ANALYSIS OF CHROMOSOMES OF

DIFFERENT ORGANISMS - REVEALED THAT
CHROMOSOMES CONTAIN PROTEINS AND NUCLEIC ACIDS
(DNA AND RNA).
 TETRANUCLEOTIDE HYPOTHESIS
INFORMATIONAL ROLES OF GENES → CHROMOSOMAL
PROTEINS

NUCLEIC ACIDS TOO SIMPLE TO CARRY GENETIC

INFORMATIONS
CONTROVERSY - 1949
 A. MIRSKY & H. RIS
ALL CELLS OF AN ORGANISM APPEARED TO CONTAIN THE SAME AMOUNT
OF DNA, FAVOURING
DNA AS THE GENETIC MATERIAL.

1953 → UNIVERSALLY ACCEPTED THAT DNA IS THE GENETIC

SUBSTANCE (i.e, CHEMICAL OF WHICH GENES ARE
COMPOSED) OF MOST MICROORGANISMS AND HIGHER
ORGANISMS.
LATER ON, RNA WAS FOUND TO BE THE GENETIC MATERIAL
OF SOME VIRUSES
DNA STRUCTURE
 RNA
STRUCTURE
Proper exploiting this molecular technology to
explore the composition of endodontics
microbiota provides valuable information
regarding identification and better
understanding of causative factor to enhance
high success rate in endodontic treatment.
 MICROORGANISMS

PROKARYOTES EUKARYOTES

Does not have membrane

bonded nuclei Membrane bounded
No mitochondria & golgi nucleus
complex present. Ex. Fungi
Eg. Bacteria and archaeal
cells
 30 S subunit
- 16S rRNA
molecules
approximatel
These cells y 1540
have 70 S nucleotides
Ribosome
composed of
30S & 50S 50S subunit -
23S rRNA
molecules
approximately
2900
nucleotides
 40s subunit
-18Sr RNA
60s subunit -
Cells have 80 25Sr RNA -
S ribosomes 5.8Sr
SmallRNA
subunit
composed genes 16Sr
40s subunit DNA & 18S
and 60s rDNA
subunit Larger
subunit genes
23Sr DNA &
25Sr DNA
have been
widely used
microbial
identification.
• Universally
distributed
among bacteria
• Long enough to
be highly
informative and
short enough to • Possesses
be easily conserved and
sequenced variable regions,
and
• Affords
16S rRNA gene reliability for
or inferring
16S rDNA phylogenetic
relationships
 There are a plethora of molecular methods for the study of
microorganisms,
 Broad-range PCR followed by cloning and sequencing can be
used to unravel the breadth of microbial diversity in a given
environment.
 Microbial community structures can be analyzed via
fingerprinting techniques, such as denaturing gradient gel
electrophoresis (DGGE) and terminal restriction fragment length
polymorphism (T-RFLP).
 Fluorescence in situ hybridization (FISH) can measure
abundance of target species and provide information on their
spatial distribution in tissues.
 Among other applications, DNA-DNA hybridization arrays,
species-specific PCR, nested PCR, multiplex PCR, and
quantitative real-time PCR can be used to survey large
numbers of clinical samples for the presence of target species.
 Variations in PCR technology can also be used to type
microbial strains.
 HYBRIDIZATIO

MOLECULAR
METHODS
N

AMPLIFICATIO
N

SEQUENCING
 PCR (or) MOLECULAR
PHOTOCOPYING

PCR method is based on the

in vitro replication of DNA
through repetitive cycles of
 Denaturation
 Primer annealing, and
 Extension steps
carried out in automated
devices called
thermocyclers.
 The target DNA serving as template denatures at high
temperatures generating single strands of DNA.

The temperature then decreases so that two short oligonucleotide

primers can anneal to their complementary sequences on opposite
strands of the target DNA.

Primers are selected to encompass the desired genetic

material, flanking the ends of the stretch of DNA to be
The result is an exponential amplification of the DNA
copied
fragment
In sequence,flanked by the second
a complementary primers, which
strand of newconfers
DNA is
TAQ extraordinary sensitivity
synthesized through in detecting
the extension the target
of each annealed DNA.
primer by a
POLYMERASE thermostable DNA polymerase in the presence of excess
deoxyribonucleoside triphosphates.

All previously synthesized products act as templates for new

primer-extension reactions in each ensuing cycle.
 Cultureusing non-selective media can detect 104
to 105 cells in a sample, and when selective
media are used, the sensitivity of culture method
increases to 103 cells.
 PCR methodology is at least 10 to 100 times more
sensitive than the other more sensitive
identification method.
 Touchdown PCR
A strategy to increase the
specificity of the assay.
 The annealing temperature
in the initial PCR cycle is
set several degrees above
the Tm of the primers.
 In subsequent cycles, the
annealing temperature is
decreased in steps of 0.5 to
2ºC per cycle until a
temperature is reached
that is equal to, or 2 to 5ºC
below, the Tm of the
primers.
 Touchdown techniques have been considered
useful to avoid the amplification of spurious DNA
fragments (non-target gene fragments and/or
fragments with improper sizes).
 Nested PCR
 Nested PCR consists of two
rounds of amplification
using different sets of
primers in each round.
 A target region of DNA is
amplified with an outer
primer pair in an initial
reaction, followed by a
second amplification using
an internal primer pair.
 Devised mainly to have
increased sensitivity, but
can also exhibit increased
specificity.
 Multiplex PCR
 In multiplex PCR, two or
more sets of primers
specific for different
targets are concomitantly
used in the same reaction.
 Thus, contrary to
conventional PCR
approaches, which can
detect only one target at a
time, multiplex PCR assays
permit the simultaneous
detection of different
species in a sample.
 Reverse
Transcriptase PCR
 Reverse transcriptase PCR
(RT-PCR) was developed to
amplify RNA targets and to
exploit the use of the
enzyme reverse
transcriptase, which can
synthesize a strand of
complementary DNA
(cDNA) from an RNA
template.
 Most RT-PCR assays
employ a two-step
approach.
 Reverse
Transcriptase PCR
 In the first step, reverse
transcriptase converts RNA
into single-stranded cDNA.
 In the second step, PCR
primers, DNA polymerase,
and nucleotides are added
to create the second
strand of cDNA.
 Once the double-stranded
DNA template is formed, it
can be used as template
for amplification as in
conventional PCR.
 The RT-PCR process may be modified into a one-
step approach by using it directly with RNA as the
template.
 In this approach, an enzyme with both reverse
transcriptase and DNA polymerase activities is
used, such as that from the bacteria Thermus
thermophilus (Tth).
 Real-Time PCR
 PCR assays are usually
qualitative or can be
adjusted to be semi-
quantitative.
 One exception is the
realtime PCR, which allows
the quantification of the
amount of DNA in the
sample by monitoring the
release of fluorescence
with each amplification
cycle.
 Real-Time PCR
 Accumulation of PCR
products is measured
automatically during each
cycle in a closed tube
format using a thermocycler
combined with a
fluorimeter.
 Realtime PCR assays allow
the quantification of
individual target species as
well as total bacteria in
clinical samples.
Advantages
 Rapidity of the assay (30 to 40 minutes), the ability to
quantify and identify PCR products directly without the
use of agarose gels,
 Contamination can be limited due to avoidance of
postamplification manipulation.
 There are several different real-time PCR approaches,
but the most commonly used chemistries include SYBR-
Green, TaqMan, and molecular beacons.
 Broad-Range PCR
 Used to investigate the
breadth of microbial diversity
in a given environment.
 Primers are designed that are
complementary to conserved
regions of a particular gene.
 Primers that are
complementary to conserved
regions of the 16S rRNA gene
have been used with the
intention of exploiting the
variable internal regions of
amplified sequence for
sequencing and further
identification.
 Initially,bacterial DNA is extracted directly from
samples, and the 16S rRNA gene is isolated via
PCR amplification with oligonucleotide primers
specific for conserved regions of the gene
(universal or broad-range primers).
 Amplification with universal primers results in a
mixture of the 16S rRNA genes amplified from
virtually all bacteria present in the sample.
 Inmixed infections, direct sequencing of the PCR
products cannot be performed because there are mixed
products from the different species composing the
consortium.
 PCR products are then cloned into a plasmid vector,
which is used to transform Escherichia coli cells,
establishing a clone library of 16S rRNA gene from the
sample.
 Cloned genes are then sequenced individually.
 Phylogenetic analysis should also be accomplished for
accurate identification.
 ADVANTAGES
Detect the unexpected bacterial diversity in a sample, it is far
more effective and accurate than culture.

Identification of several novel fastidious or as-yet-uncultivated

bacterial pathogens directly from diverse human sites.

Broad-range PCR products from samples can be alternatively

analyzed by fingerprinting techniques, such as DGGE and T-RFLP.

Genetic fingerprinting techniques can be used to determine the

structure and diversity of microbial communities living in a given
environment and to monitor changes in the community
Limitations of PCR- derived technologies
 a)Most PCR assays used for identification
purposes qualitatively detect the target
microorganism but not its levels in the sample.
Quantitative results can however be obtained in
real-time PCR assays.
 b) Most PCR assays only detect one species or a
few different species (multiplex PCR) at a time.
However, broad-range PCR analysis can provide
information about the identity of virtually all
species in a community.
c) Like DNA-DNA hybridization, most PCR assays only
detect target species and consequently fail to
detect unexpected species. This can be overcome
by broad-range PCR assays.
d) In addition to being laborious and costly, broad-
range PCR analyses can be affected by some factors,
such as biases in homogenization procedures,
preferential DNA amplification and differential DNA
extraction.
e) Microorganisms with thick cell walls, such as
fungi, may be difficult to break open and may
require additional steps for lysis and consequent
DNA release to occur.
f) False positive results have the potential to occur
because of PCR amplification of contaminant DNA.
The most important means of contamination is
through carryover of amplification product and
special precautions should be taken to avoid this
g) False negatives may occur because of enzyme
inhibitors or nucleases present in clinical samples,
which may abort the amplification reaction and
degrade the DNA template, respectively. Analysis of
small sample volumes may also lead to false
negative results, particularly if the target species is
present in low numbers.
 DGGE
 The analysis of broad-range PCR products by DGGE is a
useful strategy to fingerprint bacterial communities.
 In DGGE, DNA fragments of the same length but with
different nucleotide sequences can be separated in
polyacrylamide gels containing a linearly increasing
gradient of DNA denaturants.
 As the PCR product migrates in the gel, it encounters
increasing concentrations of denaturants and becomes
partially or fully denatured.
 DNA fragments with different sequences may have a
different melting behaviour and will stop migrating at
different positions in the gel.
 Therefore, when PCR products from mixed microbial
communities are subjected to DGGE, the result is a
fingerprint with several different bands where, at
least theoretically, each band corresponds to a single
species.
 In DGGE, multiple samples can be analyzed
concurrently, making it possible to compare the
structure of the microbial community of different
samples and to follow changes in microbial populations
over time, including after antimicrobial treatment.
 Specific bands can also be excised from the gels, re-
amplified, and sequenced to allow species
identification.
 T-RFLP →Terminal restriction
fragment length polymorphism
 T-RFLP can also provide
insight into the
structure and function
of bacterial
communities.
 In T-RFLP, the 16S rRNA
gene from different
bacterial species in a
community is PCR
amplified using one of
the universal PCR
primers labeled with a
fluorescent dye.
 T-RFLP →Terminal restriction
fragment length polymorphism

 The mixture of PCR products

is then digested with one or
more restriction enzymes
that have four basepair
recognition sites, generating
different fluorescently
labeled terminal fragment
lengths, whose sizes and
relative abundances are
determined using an
automated DNA sequencer.
 The use of a fluorescently labeled primer limits
the analysis to only the terminal fragments of the
enzymatic digestion.
 Allterminal fragment sizes generated from
digestion of PCR products can be compared with
the terminal fragments derived from sequence
databases in order to infer species identification.
 Through application of automated DNA sequencer
technology, T-RFLP has considerably greater
resolution than DGGE.
 DNA-DNA
HYBRIDIZATION
 DNA-DNA hybridization
methodology is the process
of annealing the
complementary bases of 2
single stranded DNA
molecules.
 Itemploys labeled DNA
probes that can locate and
bind to a target sequence,
forming a new duplex
molecule. The labeled
duplex can then be
detected.
 FISH – Fluorescence In
Situ Hybridization
 This method uses
fluorescently labeled rRNA
probes and fluorescence
microscopy to detect intact
microbial cells directly in
clinical specimens.
 In addition to provide
identification, FISH gives
information about presence,
morphology, number,
organization, and spatial
distribution of
microorganisms
A typical FISH protocol
includes four steps:
 Fixation and
permeabilization of the
sample;
 Hybridization with the
respective probes for
detecting the respective
target sequences;
 Washing steps to remove
unbound probe; and
 Detection of labeled cells
by microscopy or flow
cytometry.
 MOLECULAR BIOLOGICAL METHODS
ADVANTAGES LIMITATIONS
Detect both cultivable and as- Most assays are qualitative or
yet-uncultivated species or semi-quantitative (exceptions:
strains real-time PCR, microarray)
High specificity and accurate Most assays only detect one
identification of strains with species or a few different species
ambiguous phenotypic behavior at a time (exceptions: broad-
range PCR, checkerboard,
microarray)
Detect species directly in clinical Most assays detect only the
samples target species and fail to detect
unexpected species (exception:
 ADVANTAGES
High sensitivity
Rapid—most assays take no
more than minutes to a few
hours to identify a microbial
species
Do not require carefully
controlled anaerobic conditions
during sampling and
transportation
Can be used during
LIMITATIONS
Some assays can be laborious
and costly (eg., broad-range
PCR)
Biases in broad-range PCR
introduced by homogenization
procedures, preferential DNA
amplification, and differential
DNA extraction
Hybridization assays using whole
genome probes detect only
cultivable species
Can be very expensive
 ADVANTAGES LIMITATIONS
Anaerobic handling and Detect dead microorganisms
expertise not require
Samples can be stored
frozen for later analysis
DNA can be transported
easily between laboratories
Detect dead microorganisms
 CONCLUSION

• the introduction of molecular approaches in oral

microbiologyresearchhasbroughtaboutasignificantbodyofnew
knowledge with regard to the oral microbiota in health and
disease. The
developmentofmolecularbacterialidentificationmethodshasm
adeitpossible to study the role that uncultivable bacteria or
even other micro-
organismssuchasarchaeaplayinoraldiseases(133–
137).Consequently,a
significantrevolutionintheknowledgeoftheoralmicrobiotainhea
lth
 • INGLE’S ENDODONTICS 6TH EDITION
• MOLECULAR BIOLOGY 5TH EDITION – F.
WEAVER
• CELL BIOLOGY, GENETICS, MOLECULAR
BIOLOGY, EVOLUTION AND ECOLOGY 14TH
EDITION - P.S. VERMA
• MOLECULAR DIAGNOSTIC METHODS IN
ENDODONTICS – NAYAK
Works • DENATURING GRADIENT GEL
cited ELECTROPHORESIS (DGGE) FIONA
STRATHDEE AND ANDREW FREE – 2013
• EXPLOITING MOLECULAR METHODS TO
EXPLORE ENDODONTIC INFECTIONS:
PART 1—CURRENT MOLECULAR
TECHNOLOGIES FOR MICROBIOLOGICAL
DIAGNOSIS - J. F. SIQUEIRA - 2005