molecular diagnostics. the success of modern medicine and agriculture depends on the detection of...
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Molecular DiagnosticsThe success of modern medicine and
agriculture depends on the detection of specific molecules e.g.
VirusesBacteriaFungiParasitesProteins and Small Molecules
In water, plants, soil and humans.2
Characteristics of a Detection SystemA 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 ProceduresImmunological diagnostic procedures are often
used to:♠ Test drugs♠ Monitor cancers♠ Detect pathogensLimitation if the target is protein part ( biochemical)
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.5
ELISAAddition 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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Antigens and AntibodiesMost antigens have many
antigenic determinants or epitopes
Injecting antigens into mammal produces serum of polyclonal antibodies Bind to many different
epitopes and with different affinities
A uniform, high affinity, highly specific antibody preparation would be preferable
Monoclonal antibodies
Drawbacks of Polyclonal Abs
In diagnostics:1.The amount of different Abs within a polyclonal preparation may be very from one batch to the next
2.they cant be used to distinguish between two similar targets
e.g. when the difference b.w the pathogenic (target) and Non Pathogenic one (non target) is single determined.
Monoclonal AntibodiesB Cell can’t be culture but their hybrid can.
Each B cell produces only one antibody (all of the molecules produced are identical)
A clone of B cells therefore all produce identical antibodies
Identify the proper clone and the culture of these cells produces a monoclonal antibody against the desired epitope
HAT SelectionCells obtain purines and pyrimidines by either of
two waysDe novo biosynthesis requiring dihydrofolate
reductase (DHFR) activity
Salvage requiring HGPRTase (hypoxanthine guanine phosphoribosyl transferase) for purines
Drug aminopterin inhibits DHFR forcing cells to utilize salvage pathways
HGPRTase (-) cells cannot salvage purines (hypoxanthine or guanine)
HAT Selection ProcedureHAT
Hypoxanthine Aminopterin Thymidine
Protocol Immunize mouse
with Ag Isolate spleen (B
cells) Fuse with HGPRT-
myeloma tumor cells Select for actively
dividing HGPRT+ cells on HAT medium
Screening for Monoclonal Antibody Production
HGPRT+ clones screened by immunoassay (use in ELISA protocol)
Clones that produce antibody binding to target antigen cultured and further characterized
DNA Diagnostic SystemsPresence of an organisms genetic material is a
strong indicator of the presence of the organism or infectious agent
DNA Diagnostic Systems include:DNA HybridizationPCRRestriction endonuclease analysisRAPD (random amplified
polymorphic DNA)DNA fingerprinting
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DNA HybridizationBacterial 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.
Nucleic Acid Hybridization diagnostic test has 3 critical elements: Probe DNA, Target DNA, Signal detection.
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DNA HybridizationFor 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 Diagnostic Probes
Useful for detection of parasites Distinguish readily
between subtypes Distinguish readily
between present and past infections (not necessarily easily done by some ELISA protocols)
PCR makes highly sensitive
DNA or RNA: long (>100 nts) or short (<50 nts): and chemically synthesis, Cloned intact gene, or isolated region of a gene
Nonradioactive Detection Procedures
Chemiluminescent detection of bound DNA probe Biotin-labeled
nucleotides Streptavidin (SA)
binds biotin Alkaline phosphatase
conjugated to biotin also binds SA
AP cleaves small molecule releasing light
Detection by Fluorescent Dyes
Fluorescent dye attached to PCR primer(s)
PCR product now fluorescent-labeled
Detect following laser activation
Molecular Beacons
Probe Palindromic region Fluorophore Quencher
Binding of probe to target sequence separates quencher from fluorophore
Laser activation for detection
Molecular Beacons
Molecular beacons with different fluorophores allow for simultaneous testing for multiple sequences
Detection of MalariaMalaria is caused by the parasite Plasmodium
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.
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Detection of MalariaA DNA diagnostic system would only measure
current infection.
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 MalariaProbes 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) (188bp DNA
present in multiple copies)
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Polymerase Chain ReactionPCR 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 HIVRT-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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Using PCR to Detect for HIVSpecific 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.
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DNA Fingerprinting (RFLP)RFLP = Restriction Fragment Length
PolymorphismRegular 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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Minisatellite DNAAfter hybridization the membranes are stripped
and reprobed.
The probes used are human minisatellite 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. 34
Minisatellite DNAThese 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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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. 37
RAPDThe 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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Bacterial BiosensorsBacterial sensors can be used to test for
environmental pollutants.
Bacteria with bioluminescent are good candidates for pollutant sensors.
In the presence of pollutants the bioluminescent decreases.
The structural genes (luxCDABD) (vibrio fischeri) encodes the enzyme for bioluminescent was cloned into the soil bacteria Pseudomonas fluorescens.
The cells that luminescence to the greatest extent and grew as well as the wild type were tested as pollutant sensors.
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Bacterial BiosensorsTo screen water samples for pollutants (metal or
organic) a suspension of P. fluorescens was mixed with the solution to be tested.
After a 15 min incubation the luminescence of the suspension was measured in luminometer.
When the solution contained low to moderate levels of pollutants the bioluminescence was inhibited.
The procedure is rapid, simple, cheap and a good screen for pollutants.
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Restriction Digest Analysis(Molecular Diagnosis of Genetic disease)
Diagnosis of sickle cell anemia.
Sickle cell anemia is a genetic disease which is 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 the red blood cells are irregularly shaped.
The disease results in progressive anemia and damage to heart, lung, brain, joints and other organ systems.
This occurs because the mutant hemoglobin is unable to carry enough oxygen to supply these systems.
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Diagnosis of Sickle Cell AnemiaThe single mutation in hemoglobin cause a change
in 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)
Two primers which flank the mutant region of the globin gene is used during PCR to amplify this region of the gene.
The PCR products is digested with CvnI and separated by agarose gel electrophoresis.
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Cystic Fibrosis:Autosomal recessive Common in Europe,
500 mutation CFTR gene: (Cystic Fibrosis transmembrane conductance regulator)
Four the most common 81%.
PCR/OLALike sickle cell anemia many genetic diseases
are caused by mutant genes.
Many diseases are 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.
2 short oligonucleotides (oligo) are synthesized
Oligo 1 (probe x) is complementary to the wild type has A at 106 (3’ end). 48
PCR/OLAOligo 2 ( probe y) has G at 107 (5’ end).
The two probes are incubated with the 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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PCR/OLADNA ligase is added. The two probes will
only ligate if the two probes are perfectly aligned (as in the wild type).
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 Y is labeled at 3’ end with digoxygenin. 51
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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Padlock ProbesProbe (DNA/RNA)
complementary to target only at ends (3 and 5)
Bind probe
Ligate (if perfect match)
Ligated form remains attached to target which is attached to matrix (96-well plate)