Fundamentals of Biotechnology

Molecular Diagnostics

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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Comparison of Methods Used to Diagnose Parasite Infections

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

Targets for Monoclonal Antibody-based

Diagnostic Tests

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 Heterozygotes

Use of multiple fluorophores and lasers

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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RT-PCR Diagnosis of HIV

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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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RAPD Analyses

RAPD

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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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Bacterial Bisensor

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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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Detection of Sickle cell anemia by PCR

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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/OLA

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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)

PCR Using Florescence-labeled Primers

Primer ends at SNP locus to be analyzed

Mismatch gives no PCR product

Perfect match gives florescent product