Download diagnostics and biomarkers

DIAGNOSTICS AND BIOMARKERS
MODERN TECHNOLOGIES
FOR DIAGNOSTICS
• TYPES
• ANALYTICAL PARAMETERS
• USE PARAMETERS
•
TYPES BASED ON
• http://www.youtube.com/watch?v=QoK2xBn9
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• http://www.youtube.com/watch?v=QoK2xBn9
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Molecular Diagnostics
• The success of modern medicine depends on
the detection of specific molecules e.g.
Viruses
Bacteria
Fungi
Parasites
Proteins
• In water, plants, soil and humans.
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Characteristics of a Detection System
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A good detection system should have 3 qualities:
Sensitivity
Specificity
Simplicity
Sensitivity means that the test must be able to
detect very small amounts of target even in the
presence of other molecules.
• Specificity: the test yields a positive result for the
target molecule only.
• Simplicity: the test must be able to run efficiently
and inexpensively on a routine basis.
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Immunological Diagnostic Procedures
• Immunological diagnostic procedures are often used
to:
♠ Test drugs
♠ Monitor cancers
♠ Detect pathogens
• ELISA (Enzyme Linked Immunosorbent Assay)
• This involves the reaction of an antibody with an
antigen and a detection system to determine if a
reaction has occurred.
• ELISA involves:
• Binding of the test molecule or organism to a solid
support e.g. micro titer plate.
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ELISA
• Addition of a specific antibody (primary antibody)
which will bind to the test molecule if it is present.
• Washing to remove unbound molecules.
• Addition of secondary antibody which will bind to
the primary antibody.
• The secondary antibody usually has attached to it an
enzyme e.g. alkaline phosphatase.
• Wash to remove unbound antibody.
• Addition of a colourless substrate which will react
with the secondary antibody to give a colour reaction
which indicates a positive result.
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ELISA
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ELISA Animation
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ELISA Lab/Detection of HIV
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Detection of HIV
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DNA Diagnostic Systems
• DNA Diagnostic Systems include:
DNA Hybridization
PCR
Restriction endonuclease analysis
RAPD (random amplified polymorphic
DNA)
DNA fingerprinting
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DNA Hybridization
• Bacterial and viral pathogens may be
pathogenic because of the presence of specific
genes or sets of genes.
• Genetic diseases often are due to mutations or
absence of particular gene or genes.
• These genes (DNA) can be used as diagnostic
tools.
• This involves using a DNA probe during DNA
hybridization.
•
•
What is a DNA probe?
How does DNA hybridization work?
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DNA Hybridization
• For DNA hybridization:
• A probe is needed which will anneal to the target
nucleic acid.
• Attach the target to a solid matrix e.g. membrane.
• Denaturation of both the probe and target.
• Add the denatured probe in a solution to the target.
• If there is sequence homology between the target and
the probe, the probe will hybridize or anneal to the
target.
• Detection of the hybridized probe e.g. by
autoradiography, chemiluminsence or colorimetric.
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DNA hybridization movie
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Detection of Malaria
• Malaria is caused by the parasite Plasmodium
falciparum.
•
What kind of organism is P. falciparum?
• The parasite infects and destroys red blood cells.
• Symptoms include fever, rashes and damage to brain,
kidney and other organs.
• Current treatment involves microscopic observations of
blood smears, which is labour intensive.
• Other methods e.g ELISA does not differentiate
between past and present infection.
• Why?
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Detection of Malaria
• A DNA diagnostic system would only measure
current infection. (Why?)
• The procedure involves:
• A genomic library of the parasite was screened
with probes for parasitic DNA.
• The probes which hybridized strongly were
tested further.
• The probes were tested for their ability to
hybridize to other Plasmodium species which
do not cause malaria and to human DNA.
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Detection of Malaria
• Probes which hybridized to P. falciparum only
could be used as a diagnostic tool.
• The probe was able to detect 10 pg of purified
DNA or 1 ng of DNA in blood smear.
• Other DNA probes were developed for the
following diseases:
• Salmonella typhi (food poisoning)
• E. coli (gastroenteritis)
• Trypanosoma cruzi (chagas’ disease)
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Polymerase Chain Reaction
• PCR uses 2 sequence specific oligionucleotide
primers to amplify the target DNA.
• The presence of the appropriate amplified size
fragment confirms the presence of the target.
• Specific primers are now available for the
detection of many pathogens including bacteria
(E. coli, M. tuberculosis), viruses (HIV) and fungi.
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Using PCR to Detect for HIV
• RT-PCR (reverse transcriptase PCR).
• HIV has a ssRNA genome.
• Lyse plasma cells from the potentially infected
person to release HIV RNA genome.
• The RNA is precipitated using isoproponal.
• Reverse transciptase is used to make a cDNA
copy of the RNA of the virus.
• This cDNA is used as a template to make
dsDNA.
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RT-PCR Diagnosis of HIV
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Using PCR to Detect for HIV
• Specific primers are used to amplify a 156 bp
portion of the HIV gag gene.
• Using standards the amount of PCR product
can be used to determine the viral load.
• PCR can also be used as a prognostic tool to
determine viral load.
• This method can also be used to determine
the effectiveness antiviral therapy.
• (Brock Biology of Microorganisms 9th ed. pg
883-886).
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DNA Fingerprinting (RFLP)
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RFLP = Restriction Fragment Length Polymorphism
Regular fingerprinting analyses phenotypic traits.
DNA fingerprinting analyses genotypic traits.
DNA fingerprinting (DNA typing) is used to
characterize biological samples e.g.
In legal proceedings to identify suspects and
clear others.
Paternity testing
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DNA Fingerprinting (RFLP)
• The procedure involves:
• Collection of sample e.g. hair, blood, semen, and skin.
• Examination of sample to determine if there is enough
DNA for the test.
• The DNA is digested with restriction enzymes.
• Digested DNA is separated by agarose gel
electrophoresis.
• DNA is transferred by Southern blotting to a
membrane.
• Membrane is hybridized with 4-5 different probes.
• Detection of hybridization.
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Microsatellite DNA
• After hybridization the membranes are stripped and
reprobed.
• The probes used are human microsatellite DNA.
• These sequences occur in the human genome as
repeated sequences.
• E.g ATTAG….ATTAG….ATTAG….
• The length of the repeat is 9-40 bases occurring 10-30
times.
• The microsatellites have different length and numbers
in different individuals.
• The variability is due to either a gain or lost of repeats
during replication.
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Microsatellite DNA
• These changes do not have any biological
effect because the sequences do not code for
any protein.
• An individual inherit one microsatellite from
each parent.
• The chance of finding two individuals within
the same population with the same DNA
fingerprint is one in 105 - 108.
• In other words an individuals DNA fingerprint
is almost as unique as his or her fingerprint.
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DNA
Fingerprinting
 Scenario 1: Does the
blood on the defendant’s
shirt support his/her
involvement or innocence?
Why?
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DNA
Fingerprinting
Scenario 2 - presented in
class:
1.Under victim’s nails there
is blood and dead skin
cells.
2.Draw what the DNA
fingerprint evidence would
look like if the defendant
was guilty or innocent.
Why?
3.What issues must the
prosecutor consider?
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Random Amplified Polymorphic DNA (RAPD)
• Another method widely used in
characterization of DNA is RAPD.
• RAPD is often used to show relatedness among
DNA populations.
• In this procedure arbitrary (random) primers
are used during PCR to produce a fingerprint of
the DNA.
• A single primer is used which must anneal in 2
places on the DNA template and region
between the primers will be amplified.
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RAPD
• The primers are likely to anneal in many places on the
template DNA and will produce a variety of sizes of
amplified products.
• Amplified products are separated by agarose gel
electrophoresis and visualized.
• If the samples have similar genetic make up then the
pattern of bands on the gel will be similar and vice
versa.
• This procedure is widely used to differentiate between
different cultivars/varieties of the same plant.
• Issues to consider when using this procedure include
reproducibility, quality of DNA, and several primers
may have to be used.
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RAPD
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• The example is for plants. What might be an
example for people?
Bacterial Biosensors - Environment
• Bacterial sensors can be used to test for
environmental pollutants
• Structural genes (luxCDABD) encode enzyme for
bioluminescence were cloned into soil bacteria
Pseudomonas fluorescens.
• Cells that luminescence to the greatest extent
and grew as well as the wild type were tested as
pollutant sensors
• Bacteria with bioluminescent marker are
candidates for pollutant sensors
• In the presence of pollutants the
bioluminescence decreases
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Bacterial
Biosensor
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Bacterial Biosensors
• To screen water samples for pollutants (metal or
organic) mix suspension of P. fluorescens with
water sample
• After 15 min incubation, measure luminescence of
the suspension
• Procedure is rapid, simple, cheap possibly a good
screen for pollutants
 Does this procedure work to identify specific
pollutants?
 What additional experiments need to be done to
make this widely applicable?
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Restriction Digest Analysis
• EXAMPLE: Diagnosis of sickle cell anemia
• Sickle cell anemia is a genetic disease caused by a
single nucleotide change in the 6th aa of the  chain of
hemoglobin
• A (normal) glutamic acid and S (sickle) valine
• In the homozygous state SS red blood cells are
irregularly shaped
• Result = progressive anemia and damage to heart,
lung, brain, joints and other organ systems
• Mutant can’t carry enough oxygen to supply these
systems
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Diagnosis of Sickle Cell Anemia
• The single mutation in hemoglobin changes the
restriction pattern of the  globin gene abolishing a
CvnI site.
• CvnI site CCTNAGG (N = any nt)
• Normal DNA sequence CCTGAGG (A)
• Mutant DNA sequence CCTGTGG (S)
• Use two primers which flank mutant region of 
globin gene during PCR to amplify this
• Digest PCR products CvnI and separate them using
agarose gel electrophoresis
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• sC
Detection of
Sickle cell
anemia by PCR
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PCR/OLA
• Like sickle cell anemia many genetic diseases are
caused by mutant genes
• Many caused by a single nucleotide (nt) change in the
wild type gene
• A single nt change can be detected by PCR/OLA (
oligonucleotide ligation assay)
• E.g. The normal gene has A at nt position 106 and
mutant has a G
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PCR/OLA
• Synthesize 2 short oligonucleotides (oligos)
– Oligo 1 (probe x) complementary to wild type
having A at 106 (3’ end)
– Oligo 2 (probe y) has G at 107 (5’ end)
• Label them differently
• Incubate both probes with PCR amplified target
DNA
• Add DNA ligate: two probes will only ligate if
the two probes are perfectly aligned (as in the
wild type).
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PCR/OLA
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PCR/OLA
• To determine if the mutant or wild type
gene is present it is necessary to detect for
ligation.
• Probe x is labeled at 5’ end with biotin
• Probe x is labeled at 5’ end with
digoxygenin.
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PCR/OLA
• Digoxygenin serves as an antibody binding
indicator.
• After washing a colourless substrate is
added.
• If a coloured substrate appears this is
indicative that the biotin probe (x) ligated
to the dioxygenin probe (Y) and that the
wild type gene is present.
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PCR/OLA
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PCR/OLA
• Synthesize 2 short oligonucleotides (oligos)
– Oligo 1 (probe x) complementary to wild type having A
at 106 (3’ end).
– Oligo 2 (probe y) has G at 107 (5’ end).
• Incubate both probes with PCR amplified target
DNA
• For the wild type the two probes anneal so that
the 3’end of probe x is next to the 5’end of probe y.
• For the mutant gene the nt at the 3’ end of probe x
is a mismatch and does not anneal.
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END