Download Modern methods in Molecular Pathology

Presented By:
Safia Nisar
Sumaiya Gul
Increasing Knowledge of Molecular
basis of the disease
Advances in technology for
analyzing nucleic acids and gene
products
Recent introduction of array
technology help to analyze multiple
genes, transcripts and Proteins
• A pathologist needs to have adequate
Knowledge of
Molecular
Biology
Human
Genetics
Microbiology
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Infectious Disease Testing
Molecular Oncology
Molecular Genetics
Histocompatibilty
• Performed most frequently
• Quantification of Infectious agents
• Identification of difficult or impossible to
cultivate agents
• Identification of antiviral or antibiotic
resistance genes
• Identification of toxin genes
• Advances in the Knowledge of biology of
leukemias and lymphomas
• Understanding the molecular genetics of solid
tumors
• Genetic alterations associated with the
occurrence of neoplasia
• Sub-discipline of Medical genetics
• Involves discovery and Laboratory testing for
DNA that underlie single gene disorders
• May also be used in the diagnosis of
syndromes involving
epigenetic abnormalities
Achondroplasia
Cystic Fibrosis
Huntington
Disease
Duchene
Muscular
distrophy
Hereditary
Breast Cancer
Angleman
Syndrome
BeckwithWiedeman
syndrome
Prader-willi
Syndrome
Uniparental
disomy
• Transformation from Serologic testing to DNA
based identification
• More precision and accuracy
• Sequence specific PCR
• Sequence specific oligonucleotide
hybridization
• DNA sequencing of PCR products
• Routine procedure to collect DNA for
molecular or Forensic analysis
• Steps Involved
Cell Lysis
Removal of
membrane
lipids
Removal of
Proteins
Removal of
RNA
Precipitation
of DNA by
alcohol
• Purification of RNA
• Somewhat complicated
• Guanidinium thiocyanate-phenol-chloroform
extraction
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PCR (Polymerase Chain Reaction)
LCR (Ligase Chain Reaction)
SDA (Strand Displacement Amplification)
Hybridization
Diagnostic Markers
BDA (Branched DNA Assay)
TMA (Transcription Mediated Amplification)
• Makes large copies of a gene
• Generally three step; Denaturation, Anealing
and Extension
• Amount of DNA is insufficient otherwise
• Diagnosis of Disease: Linkage analysis,
detection of mutant alleles, diagnosing
infectious agents, epidemiological studies
• DNA Amplification Technique
• Amplification of the nucleic acid used as probe
• For each of the two DNA strands, two partial
probes are ligated to form the actual one; uses
DNA Polymerase and DNA Ligase
• Each cycle results in the doubling of target nucleic
acid molecule
• Greater Specifity
• Used for Single base mutations and genetic
disorders
• An isothermal nucleic acid amplification method
• Primer contains a restriction site annealed to template
• Amplification primers are then annealed to 5' adjacent
sequences (form a nick) and start amplification at a
fixed temperature
• Newly synthesized DNA are nicked by a restriction
enzyme
• Polymerase starts amplification again, displacing the
newly synthesized strands
• 109 copies of DNA can be made in one reaction
• In situ hybridization (ISH) is a powerful
technique for localizing specific nucleic acid
targets within fixed tissues and cells, allowing
you to obtain temporal and spatial
information about gene expression and
genetic loci.
• The nucleic acid probe is synthesized, labeled,
purified, and annealed with the specific target
• The difference is the greater amount of
information gained by visualizing the results
within the tissue.
• Today there are two basic ways to visualize
RNA and DNA targets in situ:
1. Fluorescence (FISH) and
2. Chromogenic (CISH) detection.
• Multiplex fluorescence in situ hybridization (FISH)
exemplifies the elegance that only fluorescence-based
strategies offer: the ability to assay multiple targets
simultaneously and visualize co-localization within a
single specimen. Using spectrally distinct fluorophore
labels for each different hybridization probe, this
approach gives you the power to resolve several
genetic elements or multiple gene expression patterns
in a single specimen, with multicolor visual display.
• A process in which a labeled complementary DNA
or RNA strand is used to localize a specific DNA or
RNA sequence in a tissue specimen. CISH
methodology may be used to evaluate gene
amplification, gene deletion, chromosome
translocation, and chromosome number.
• Also known as a biomarker, it is a measurable
characteristic that reflects the severity or
presence of some disease state. More
generally a biomarker is anything that can be
used as an indicator of a particular disease
state or some other physiological state of an
organism.
• A biomarker can be a substance that is introduced
into an organism as a means to examine organ
function or other aspects of health. For example,
rubidium chloride is used as a radioactive isotope
to evaluate perfusion of heart muscle. It can also
be a substance whose detection indicates a
particular disease state, for example, the presence
of an antibody may indicate an infection. More
specifically, a biomarker indicates a change in
expression or state of a protein that correlates
with the risk or progression of a disease, or with
the susceptibility of the disease to a given
treatment.
• Branched DNA assay is a signal amplification
assay (as opposed to a target amplification
assay) that is used to detect nucleic acid
molecules.
1. From the base up, a branched DNA assay
begins with a dish or some other solid
support (e.g., a plastic dipstick). The dish is
peppered with small, single stranded DNA
molecules (or chains) that 'stick up' into the
air. We'll call these "capture probe" DNA
molecules.
2. An "extender" DNA molecule is added. Each
"extender" has two domains, one that
hybridizes to the capture DNA molecule and
one that "hangs out" in the air. The purpose
of the extender is two-fold. First, it creates
more available surface area for target DNA
molecules to bind, and second, it allows the
assay to be easily adapted to detect a
variety of target DNA molecules.
3. Once the capture and extender molecules are
in place and they have hybridized, the sample
can be added. Target molecules in the sample
will bind to the extender molecule. So we
have a base peppered with capture probes,
which are hybridized to extender probes,
which in turn are hybridized to target
molecules.
4. At this point, signal amplification takes
place. A "label extender" DNA molecule is
added that has two domains (similar to the
first extender). The label extender hybridizes
to the target and to a "pre-amplifier"
molecule. The preamplifier molecule has two
domains. First, it binds to the label extender
and second, it binds to the amplifier
molecule. An example amplifier molecule is
an oligonucleotide chain bound to the
enzyme alkaline phosphatase.
• Transcription-Mediated Amplification (TMA) is
an RNA transcription-mediated amplification
system using two enzymes to drive the
reaction: RNA polymerase and reverse
transcriptase.
• TMA is isothermal; the entire reaction is
performed at the same temperature in a
water bath or heat block. This is in contrast to
other amplification reactions such as PCR that
require a thermal cycler instrument to rapidly
change the temperature to drive reaction.
• TMA can amplify either DNA or RNA, and
produces RNA amplicon, in contrast to most
other nucleic acid amplification methods that
only produce DNA.
• TMA has very rapid kinetics, resulting in a
billion-fold amplification with 15-60 minutes.
TMA can be combined with HPA for endpoint
detection or with molecular torches for realtime detection.
• There are no wash steps, and no amplicon is
ever transferred out of the tube, which
simplifies the procedure and reduces the
potential for contamination.