DIAGNOSTICS AND BIOMARKERS

MODERN TECHNOLOGIES FOR DIAGNOSTICS

• TYPES• ANALYTICAL PARAMETERS• USE PARAMETERS•

TYPES BASED ON

• http://www.youtube.com/watch?v=QoK2xBn9fSc

• http://www.youtube.com/watch?v=QoK2xBn9fSc

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Molecular Diagnostics

• The success of modern medicine depends on the detection of specific molecules e.g.

VirusesBacteriaFungiParasitesProteins• In water, plants, soil and humans.

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Characteristics of a Detection System

• 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 HybridizationPCRRestriction endonuclease analysisRAPD (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)

• 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

• The example is for plants. What might be an example for people?

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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 CCTNAGG (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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Detection of Sickle cell

anemia by PCR

• sC

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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.

END