9/21/2010 1 course outline molecular diagnostics. introduction, the use of elisa, dna hybridization,...

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9/21/2010 1 Course Outline Molecular diagnostics. Introduction, the use of ELISA, DNA hybridization, PCR,, RAPD and DNA fingerprinting, bacterial biosensors, PCR/OLA, and restriction digest, for DNA diagnostics. Therapeutic agents and vaccines. Enzymes as therapeutic agents, production of monoclonal antibodies in E. coli, nucleic acids as therapeutic agents, HIV therapeutic agents, subunit vaccines, attenuated vaccines, and vector vaccines. The synthesis of commercial products by recombinant micro- organisms. Production of restriction enzymes, ascorbic acid, microbial synthesis of indigo and the production of xanthan gum. *Text: Glick, B.R. and Pasternak, J.J. Molecular Biotechnology, Principles and Applications of Recombinant DNA Technology. 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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Page 1: 9/21/2010 1 Course Outline Molecular diagnostics. Introduction, the use of ELISA, DNA hybridization, PCR,, RAPD and DNA fingerprinting, bacterial biosensors,

9/21/2010

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Course OutlineMolecular diagnostics. Introduction, the use of ELISA, DNA

hybridization, PCR,, RAPD and DNA fingerprinting, bacterialbiosensors, PCR/OLA, and restriction digest, for DNA

diagnostics.Therapeutic agents and vaccines. Enzymes as therapeutic agents,

production of monoclonal antibodies in E. coli, nucleic acids astherapeutic agents, HIV therapeutic agents, subunit vaccines,

attenuated vaccines, and vector vaccines.The synthesis of commercial products by recombinant micro-

organisms. Production of restriction enzymes, ascorbic acid,microbial synthesis of indigo and the production of xanthan

gum.

*Text: Glick, B.R. and Pasternak, J.J. Molecular Biotechnology,Principles and Applications of Recombinant DNA

Technology.

Molecular Diagnostics

The success of modern medicine dependson the detection of specific molecules e.g.VirusesBacteriaFungi

ParasitesProteins

In water, plants, soil and humans.

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Characteristics of a DetectionSystem

A good detection system should have 3 qualities :SensitivitySpecificitySimplicitySensitivity means that the test must be able to

detect very small amounts of target even inthe presence of other molecules.

Specificity: the test yields a positive result forthe target molecule only.

Simplicity: the test must be able to runefficiently and inexpensively on a routinebasis.

Immunological DiagnosticProcedures

Immunological diagnostic procedures are oftenused to:

Test drugsMonitor cancersDetect pathogens

ELISA (Enzyme Linked Immunosorbent Assay)This involves the reaction of an antibody with anantigen and a detection system to determine if areaction has occurred.ELISA involves :Binding of the test molecule or organism to asolid support e.g. micro titer plate.

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ELISA

Addition of a specific antibody (primaryantibody) which will bind to the test molecule ifit is present.Washing to remove unbound molecules.Addition of secondary antibody which willbind to the primary antibody.The secondary antibody usually has attached to itan enzyme e.g. alkaline phosphatase.Wash to remove unbound antibody.Addition of a colourless substrate which willreact with the secondary antibody to give acolour reaction which indicates a positive result.

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Detection of HIV

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 pathogenicbecause of the presence of specific genes or setsof genes.Genetic diseases often are due to mutations orabsence of particular gene or genes.These genes (DNA) can be used as diagnostictools.This involves using a DNA probe during DNA

hybridization.

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 tothe target.

Detection of the hybridized probe e.g. byautoradiography, chemiluminsence or colorimetric.

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Detection of Malaria

Malaria is caused by the parasite Plasmodiumfalciparum.What kind of organism is P. falciparum?The parasite infects and destroys red blood cells.Symptoms include fever, rashes and damage tobrain, kidney and other organs.Current treatment involves microscopicobservations of blood smears, which is labourintensive.Other methods e.g ELISA does not differentiatebetween past and present infection.

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Detection of Malaria

A DNA diagnostic system would only measurecurrent infection.The procedure involves :A genomic library of the parasite was screenedwith probes for parasitic DNA.The probes which hybridized strongly weretested further.The probes were tested for their ability tohybridize to other Plasmodium species whichdo not cause malaria and to human DNA.

Detection of Malaria

Probes which hybridized to P. falciparum only couldbe used as a diagnostic tool.The probe was able to detect 10 pg of purifiedDNA or 1 ng of DNA in blood smear.Other DNA probes were developed for thefollowing diseases:Salmonella typhi (food poisoning)E. coli (gastroenteritis)Trypanosoma cruzi (chagas’ disease)

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Polymerase Chain Reaction

PCR uses 2 sequence specific oligionucleotideprimers to amplify the target DNA.

The presence of the appropriate amplified sizefragment confirms the presence of the target.

Specific primers are now available for the detectionof many pathogens including bacteria (E. coli, M.tuberculosis), viruses (HIV) and fungi.

Using PCR to Detect for HIV

RT--PCR (reverse transcriptase PCR).HIV has a ssRNA genome.Lyse plasma cells from the potentially infectedperson to release HIV RNA genome.The RNA is precipitated using isoproponal.Reverse transciptase is used to make a cDNAcopy of the RNA of the virus.This cDNA is used as a template to makedsDNA.

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RT--PCR Diagnosis of HIV

Using PCR to Detect for HIV

Specific primers are used to amplify a 156 bpportion of the HIV gag gene.Using standards the amount of PCR product canbe used to determine the viral load.PCR can also be used as a prognostic tool todetermine viral load.This method can also be used to determine theeffectiveness antiviral therapy.(Brock Biology of Microorganisms 9th ed. pg883--886).

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Restriction Digest Analysis

Diagnosis of sickle cell anemia.Sickle cell anemia is a genetic disease which iscaused by a single nucleotide change in the 6th aaof the chain of hemoglobin.A (normal) glutamic acid and S (sickle) valine.In the homozygous state SS the red blood cellsare irregularly shaped.The disease results in progressive anemia anddamage to heart, lung, brain, joints and otherorgan systems.This occurs because the mutant hemoglobin isunable to carry enough oxygen to supply thesesystems.

Diagnosis of Sickle Cell Anemia

The single mutation in hemoglobin cause achange in the restriction pattern of the globingene 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 ofthe globin gene is used during PCR to amplifythis region of the gene.The PCR products is digested with CvnI andseparated by agarose gel electrophoresis.

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Detectionof Sickle

cellanemiaby PCR

PCR/OLA

Like sickle cell anemia many genetic diseases arecaused 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 106and mutant has a G.2 short oligonucleotides (oligo) are synthesizedOligo 1 (probe x) is complementary to the wildtype has A at 106 (3’ end).

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PCR/OLA

Oligo 2 (probe y) has G at 107 (5’ end).The two probes are incubated with the PCRamplified target DNA.For the wild type the two probes anneal so thatthe 3’end of probe x is next to the 5’end of probey.For the mutant gene the nt at the 3’ end of probex is a mismatch and does not anneal.

PCR/OLA

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PCR/OLA

DNA ligase is added. The two probes will onlyligate if the two probes are perfectly aligned (as inthe wild type).To determine if the mutant or wild type gene ispresent it is necessary to detect for ligation.Probe x is labeled at 5’ end with biotinProbe y is labeled at 5’ end with digoxygenin.

PCR/OLA

Digoxygenin serves as an antibody bindingindicator.After washing a colourless substrate is added.If a coloured substrate appears this is indicativethat the biotin probe (x) ligated to the dioxygeninprobe (y) and that the wild type gene is present.

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PCR/OLA

DNA Fingerprinting (RFLP)

RFLP = Restriction Fragment LengthPolymorphismRegular fingerprinting analyses phenotypic traits.DNA fingerprinting analyses genotypic traits.DNA fingerprinting (DNA typing) is used tocharacterize biological samples.In legal proceedings to identify suspects and clearothers.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 probeDetection of hybridization.

Microsatellite DNA

After hybridization the membranes are strippedand reprobed.The probes used are human microsatelliteDNA.These sequences occur in the human genome asrepeated sequences.E.g ATTAG….ATTAG….ATTAG….The length of the repeat is 99--40 bases occurring10--30 times.The microsatellites have different length andnumbers in different individuals.The variability is due to either a gain or lost ofrepeats during replication.

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Microsatellite DNA

These changes do not have any biological effectbecause the sequences do not code for anyprotein.An individual inherit one microsatellite fromeach parent.The chance of finding two individuals within thesame population with the same DNA fingerprintis one in 105 - 108.In other words an individuals DNA fingerprintis almost as unique as his or her fingerprint.

DNAFingerprinting

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Random AmplifiedPolymorphic DNA (RAPD)

Another method widely used in characterizationof DNA is RAPD.RAPD is often used to show relatedness amongDNA populations.In this procedure arbitrary (random) primers areused during PCR to produce a fingerprint of theDNA.A single primer is used which must anneal in 2places on the DNA template and region betweenthe primers will be amplified.

RAPDThe primers are likely to anneal in many placeson the template DNA and will produce a varietyof sizes of amplified products.Amplified products are separated by agarose gelelectrophoresis and visualized.If the samples have similar genetic make up thenthe pattern of bands on the gel will be similar andvice versa.This procedure is widely used to differentiatebetween different cultivars/var of the same plant.Issues to consider when using this procedureinclude reproducibility, quality of DNA, andseveral primers may have to be used.

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RAPD

Bacterial Biosensors

Bacterial sensors can be used to test forenvironmental pollutants.Bacteria with bioluminescent are good candidatesfor pollutant sensors.In the presence of pollutants the bioluminescentdecreases.The structural genes (luxCDABD) encodes theenzyme for bioluminescent was cloned into thesoil bacteria Pseudomonas fluorescens.The cells that luminescence to the greatest extentand grew as well as the wild type were tested aspollutant sensors.

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Bacterial Biosensors

To screen water samples for pollutants (metal ororganic) a suspension of P. fluorescens was mixedwith the solution to be tested.After a 15 min incubation the luminescence of thesuspension was measured.When the solution contained low to moderatelevels of pollutants the bioluminescence wasinhibited.The procedure is rapid, simple, cheap and a goodscreen for pollutants.

BacterialBisensor