Molecular Diagnostics
Molecular Diagnostics
 Success in modern medicine and agriculture
depends on the ability to detect specific
viruses, bacteria, fungi, parasites, proteins,
and their products in the samples.
 The effective prevention, control, and
treatment of infectious diseases is facilitated
by early and accurately detection and
identification of the causative agent.
 Traditional methods for this entail the isolation
and cultivation of the agent, and analysis of
physiological properties.
 This can be time consuming, and ineffective if
the pathogen does not grow well in culture.
 Ex. Chlamydia trachomatis-common in STD-will
only grow in culture given long incubations.
 To overcome this major constraint, molecular
diagnostic procedures using immunology and
DNA technology have been devised.
 In general, these methods must be specific,
sensitive, and simple.
Immunological methods
 Used for a wide variety of applications including
drug testing, cancer detection, metabolite
identification, pathogen identification, and
monitoring infectious agents.
 Traditional pathogen identification usually
involves a process of elimination through
biochemical tests for the presence of specific
molecules.
 The uses of antibodies has revolutionized this
process.
ELISA
 Enzyme-linked immunosorbent assay.
 Uses a primary antibody against a target
antigen to be detected.
 Detection depends upon efficient binding and
substrate conversion.
 In this method, a sample(s) being tested is
coated onto a support such as a microtiter
plate.
 A blocking solution is added to prevent
background.
 A primary antibody against our antigen of interest
is added and allowed to bind, and is then washed
to remove unbound antibody.
 A secondary antibody with a bound enzyme is
added to detect the binding of the primary antibody.
 The colorless substrate for the enzyme is added,
and the absorbance of the colored product is
measured.
 Used for drug testing, monitoring cancers,
metabolites, pathogen identification, monitoring
infectious agents (human, animal, plant), etc.
 Antibodies produced by injection of an antigen
into an animal.
 The resulting antibody mixture found in the
serum are polyclonal.
 They recognize multiple antigenic epitopes on
the molecule in question.
 Most of the time these work well for ELISA.
 Two drawbacks are:(1) the amount of the
antibodies may vary from batch to batch and
(2) cross reaction may occur between the
target and nontarget.
 The surface of the antigen depicted has 7
different antigenic determinants (epitopes).
 When immunized, each epitope elicit the
synthesis of different antibody.
 Together, they constitute a polyclonal antibody.
Monoclonal Antibodies
 To produce monoclonal antibodies, a
researcher need to create a cell line that could
be grown in culture and that would produce a
single type of antibody molecule with high
affinity for a specific target antigen.
 Unfortunately, the B lymphocytes do not
reproduce in culture.
 The hybrid cell would have the B cell genetic
components to produce the antibody but can
grow in culture.
 Myeloma cells (cancerous B lymphocytes)
became candidate for cell fusion.
 To produce monoclonal antibodies, animals
are injected with the antigen, and their
spleens are collected.
 Spleens are macerated, resuspended, and
mixed with myeloma cells that have a
metabolic deficiency for the enzyme
hypoxanthine-guanine phosphoribosyl
transferase (HGPRT-).
 Polyethylene glycol is added to facilitate
fusion.
 Fused cells are grown on a selective medium
(HAT) which allow only the myeloma-spleen
fusion cells to grow.
 About 10 – 14 days post fusion treatment, the
spleen-myeloma fusion cells are then grown on
complete medium in microtiter plates.
 Fused cells must be screened for the production of
the specific antibody using the culture medium,
which contains secreted antibodies, in
immunoassay.
 The identified cells are maintained and used for
antibody production.
 Isolated antibodies are conjugated to the enzyme
for ELISA, increasing specificity since only one
epitope is recognized.
Biofluorescent and
Bioluminescent Systems
 Bioassay for potentially toxic compounds.
 The gene is placed under the promoter that
responds to certain environmental signal.
Colored Fluorescent Proteins
 Green fluorescent protein.
 The 238-amino-acid-long GFP isolated from
the jellyfish Aequorea victoria, fluoresces
green when exposed to UV light.
 The incorporation of green fluorescent protein
into cells allow intact living cells to be
monitored in real time.
 The use of this reporter molecule has
revolutionized fluorescence microscopy.
Colored Fluorescent Proteins
 Red fluorescent protein.
 Isolated from the Discosoma coral, and by
means of multiple random mutations, generated
a mutant that existed as a monomer.
 A modified monomeric red fluorescent protein
is constructed.
 The constructed then became the starting point
for several additional rounds of random
mutagenesis.
 Eventually, seven different monomeric colored
fluorescent proteins were produced.
Luciferase
 The luciferase enzyme, which catalyzes a
light-emitting reaction.
 Luciferase genes from bacteria are termed lux
genes, while those from firefly are termed luc
genes.
 The lux system produces a peak of light at 490
nm, no additional compounds need when all 5
genes are used.
 The luc system results in production of light
at 55- to 575 nm, luciferin is needed as a
substrate for the light reaction.
Microbial Biosensors
 Need for bioassay for potentially toxic
compounds in the environment.
 Often constitutively bioluminescent bacteria
are good candidates for pollutant detectors.
 Vibrio fisheri - bioluminescent marine bacteria
have limit utility because of very specific
saline and pH requirements.
 Lux genes can however be transferred to
other more useful soil bacerium, such as
Pseudomonas fluorescens.
 The engineered yeast is used to detect low levels
of estrogenic substances in the environment.
Nucleic Acid Diagnostic System
 DNA hybridization probes provide rapid and
reliable assays.
 Most often a probe is developed to recognize a
specific gene carried by a pathogen.
 Bind single-stranded DNA target to a membrane
support.
 Add single-stranded labeled DNA probe under
appropriate conditions.
 Wash the membrane to remove excess unbound
probe.
 Detect the hybrid sequences (target-probe).
Nucleic Acid Diagnostic System
 DNA probes for this process must be specific
and not recognize non-target organisms.
 Towards this end, probes usually use a gene
or gene variant specific to the organism being
investigated.
 Cross hybridization with other species must
be rigorously tested to eliminate false
positives.
 Hybridization conditions must be optimized.
Nucleic Acid Diagnostic System
 Probes for diagnosis of a variety of diseases
have been developed.
 Includes a test for the malarial parasite
Plasmodium falciparum.
 Traditional detection looks for the parasite in
blood smears which is time-consuming.
 ELISA can work, but they do not always
discriminate between current and past infection.
 Repetitive DNA probes were isolated that
specifically detect only P. falciparum.
Nucleic Acid Diagnostic System
 Over 100 such tests have been developed for
human pathogenic strains of bacteria, viruses,
and parasites.
 These include:
 Legionella pneumophila, Salmonella typhi,
Campylobacter hyointestinalis, and the
enterotoxic E. coli.
Nucleic Acid Diagnostic System
 Detection of Trypanosoma cruzi in Chagas
disease uses different technology.
 Traditional detection here uses a blood smear or
blood fed to uninfected insect vectors. Both tests
are laborious, time-consuming, and costly.
 ELISA can be used but often gives false positives.
 A set of PCR primers was developed that allows
for detection of a 188 bp fragment that is present
in mutiple copies in T. Cruzi but absent in others.
 Many pathogens are detected using the same
approach including HIV, and tuberculosis.
Nonradioactive Hybridization
 Most commonly, detection using Southern
hybridization methods uses radioactively
labeled probes.
 This has the advantage of probes that have
high specific activity.
 The disadvantages include short-lived probes,
potentially dangerous, and special handling
and disposal equipment.
 As a result, non-radioactive detection
methods have been developed.
Nonradioactive Hybridization
 One of the most common methods uses DNA
probes that have been biotinylated.
 Biotin binds very tightly with a protein from
bacterium Streptococcus called streptavidin.
 Detection often uses enzymes that are either
conjugated to streptavidin or are biotinylated
themselves.
Enzymes used
include
horseradish
peroxidase or
alkaline
phosphatase
Nonradioactive Hybridization
 Another early PCR method for detection uses
fluorescently labeled primers.
 After PCR amplification, the primers are
separated from the amplification product and
the presence of label is detected.
 If the target DNA is not present in the sample,
then no fluorescent product will be observed.
 The system is sensitive and quite rapid, since
it is not necessary to run a gel to separate the
amplify target DNA.
Molecular Beacons
 Molecular beacons are a novel non-
radioactive detection mechanism.
 The probe is 25 nucleotides long.
 The 15 nucleotides in the middle are
complementary to the target DNA.
 A fluorescent molecule (fluorophore) is
attached to 5’ end and a non-fluorescent
molecule (quencher) is attached to 3’ end.
 When hybridized, the reporter dye fluoresces,
and can be detected.
 An improvement on this is real time PCR with
the same sort of molecular beacon probes.
 Molecular-beacon
probes with
different
fluorophores that
complement to a
different target
DNA.
 Can also be
used to
distinguish
homo vs
heterozygotes.
DNA fingerprinting
 Distinguishing individuals with DNA analysis
is called DNA fingerprinting (DNA typing).
 The DNA from a sample left at the crime scene
can be analyzed and compared with the
suspects’ DNA to help in prosecution.
 It can be used to determine paternity and
identify victims from disasters.
 PCR or RFLP
DNA fingerprinting
 Probes for hybridization in forensic samples
consists of human minisatellite DNA.
 This DNA is repetitive and extensively variable
from one person to the next.
 The length of the repeats range from 9 to 40
bp, and the numbers of the repeats in the
minisatellites range from about 10 to 30.
 Southern blot of
a forensic DNA
sample.
RAPD
 Randomly Amplified Polymorphic DNA
 This technique uses PCR with random sets of
primers, 9-10 bases long.
 The primers are not directed at any particular
sequence. As short sequence, it will pair with
the chromosomal DNA at many sites.
 When the 3’ ends of the oligonucleotides on
opposite strands of the DNA face each other,
the DNA in between can be amplified.
RAPD
RAPD
 There is a number of advantages.
 The universal set of primers can be used for
all plant species.
 It is fast, easy, and repeatable.
 A large number of samples may be easily and
rapidly characterized since there is no
hybridization required.
 The process can be automated.
 It has been done successfully in many
systems.
Real-Time PCR
 By labeling the amplified DNA with a
fluorescent dye that is irradiated with light of
a certain wavelength, it is possible to watch
the production of PCR product in “real-time.”
 It is also can be used to quantify the amount
of a specific DNA fragment in the starting
material.
 Different technologies available.
 SYBR green and TaqMan.
 The SYBR green does not bind to single-
stranded DNA. Only the bound DNA
fluoresces.
 A plot of ΔRn (normalized fluorescence) versus
cycle number in real-time PCR experiment.
 Four phases of PCR are shown.
 The cycle in phase two is known as CT.
 A standard curve is first generated by serially
diluting a sample with known number of copies
of target DNA.
 The CT values for each dilution are plotted
against the starting amount of sample.
 Immunoquantitative real-time PCR
 Combines specificity of antibodies and the
sensitivity of real-time PCR.
Ancestry Determination
 Uses single nucleotide polymorphisms (SNPs)
 Commonly used autosomal DNA, the Y
chromosome, and maternal DNA
(chromosome X and mitochondrial DNA)
 Table 9.3 summarizes the current
mitochondrial DNA haplotypes and the ethnic
groups associated with these haplotypes.
 The results are often misinterpreted because
of the gene flow and the genetic diversity
within the populations.
Animal Species determination
 For endangered species and illegal trafficking
 Method uses PCR of animal’s cytochrome b
gene followed by sequencing.
Automated DNA Analysis
 It is necessary to rapidly identify the organism(s)
that is the source of infectious outbreak.
 Combination of PCR and electrospray ionization
mass spectrometry are performed.
 DNA sequences of a large numbers of microbes
have been determined.
Molecular Diagnosis of
Genetic Disease
 Previously, genetic testing relied most
exclusively on biochemical assays that
scored either the presence or absence of a
gene product.
 Test at the DNA level are definitive for
determining the existence of specific gene
mutations.
 It is possible to develop screening assays for
all single-gene diseases.
Molecular Diagnosis of
Genetic Disease
 Screening for cystic fibrosis: CF results from
mutations in cystic fibrosis transmembrane
conductance regulator (CFTR) gene, resulting
in defects in chloride ions transport.
 There are nearly 1,400 known mutations that
can cause cystic fibrosis.
 Some mutations are much more prevalent
than others (Table 9.4).
 The allele ΔF508 can be used for diagnosis.
Molecular Diagnosis of
Genetic Disease
Molecular Diagnosis of
Genetic Disease
Molecular Diagnosis of
Genetic Disease
 Sickle cell anemia: Occurs as the result of a
single nucleotide change in the codon for the
sixth amino acid of the β chain of hemoglobin
(Glu to Val.)
 Homozygous individuals develop severe
anemia resulting in progressive organ
damage as a result of an inability to carry
oxygen.
 Heterozygous individuals mostly normal
except especially under conditions of low
oxygen tension (high usage or altitude).
Molecular Diagnosis of
Genetic Disease
 Screening can be done quite simply since the
mutation destroys a restriction enzyme site.
 PCR is used to amplify the region which can
then be digested and electrophoresed.
 Normal individuals have the site and produce
a different fragment pattern than sickle cell
individuals.
Molecular Diagnosis of
Genetic Disease
 Unfortunately, not all single nucleotide
changes effect restriction sites so other types
of assays must be developed for detection.
 One of these is called PCR/OLA
(oligonucleotide ligation assay).
 First, the sequence is amplified by PCR.
 Next, a pair of oligonucleotide probes are
annealed (one is biotinylated and the other is
digoxigenated) to either side of the
susceptible site.
Molecular Diagnosis of
Genetic Disease
 DNA ligase is added. If no mutation exists,
ligation occurs, but not if there is a mutation.
 The products are then allowed to bind to a
streptavidin coated plate, and antidigoxygenin
antibody are added.
 The antibody is enzyme linked and produces a
color in presence of the substrate if bound.
 The PCR/OLA system is rapid, sensitive, and
highly specific.
Molecular Diagnosis of
Genetic Disease
 A similar technique uses a padlock probe.
 In this case, the 5’ & 3’ ends of a probe are
complimentary to the sequence and anneal in
close proximity.
 DNA ligase is added and ligation can take place.
 If there is mutation, then, there is no ligation and
hybridization.
 Unligated probe will be remove by washing.
 Ligated probe can be detected as still bound
using reporter tags or ELISA.
Molecular Diagnosis of
Genetic Disease
 Detection of a single-base mutation can also be
done by fluorescent-labeled PCR.
 Amenable to real-time PCR.
 A primer for the wild type sequence is used which
ends with at commonly mutated nucleotide.
 Wild-type DNA gives a product & mutant does not.
 The reverse assay for the mutant can also be
used with different fluorescent color.
Molecular Diagnosis of
Genetic Disease
 TaqMan Assay.
 Taqman probe, complementing to wild-type DNA
is added prior to the PCR.
 The probe contains fluorescent dye and quencher
at 5’ end and 3’ end, respectively.
 In the extension phase of PCR, the Taqman probe
is replacing by the synthesizing strand.
 Subsequently, the 5’ fluorescent dye is cleaved by
the 5’ nuclease activity of the Taq polymerase,
leading to the increase of the fluorescent dye.