M.Diagnostic 2024 850

Cytosine Adenine
Guanine
Thymine
Uracil
Molecular Diagnostics
 The success of modern medicine and agriculture
depends on the detection (identification)of specific
molecules e.g.
➢ Viruses
➢ Bacteria
➢ Fungi
➢ Parasites
➢ Proteins and Small Molecules
In water, plants, soil and humans.

15
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.16
Comparison of Methods Used to Diagnose Parasite
Infections
DNA Diagnostic Systems
 Presence of an organisms genetic material is a strong
indicator of the presence of the organism or infectious
agent
 DNA Diagnostic Systems include:

➢DNA Hybridization
➢PCR
➢Restriction endonuclease analysis
➢RAPD (random amplified polymorphic
DNA)
➢DNA fingerprinting
18
1. DNA Hybridization
Requirements:
 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.
Nucleic Acid Hybridization diagnostic test has 3 critical elements:
Probe DNA, Target DNA, Signal detection.
19
DNA Hybridization
Steps involved in 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. 20
DNA Diagnostic
Probes
Probes are short section of DNA or RNA
with an additional tagged or labeled
chemical entity that are used to bind to its
complimentary strand and thereby allows
detection of candidate nucleic acid
molecule.
 DNA or RNA: long (>100 nts) or short (<50 nts): and

chemically synthesis, Cloned intact gene, or isolated
region of a gene
 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
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
PCR Using Florescence-
labeled Primers
 Primer ends at SNP
locus to be analyzed
 Mismatch gives no PCR

product
 Perfect match gives

florescent product
3. 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.
26
Diagnosis of Sickle Cell Anemia
 The single mutation in hemoglobin cause a change in the
restriction pattern of the  globin gene abolishing a CvnI
site.
 CvnI site CCTNAGG (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.
28
Detection of
Sickle cell
anemia by PCR
29
Cystic Fibrosis:
 Autosomal recessive Common in Europe,
 1400 different types mutation CFTR gene: (Cystic

Fibrosis transmembrane conductance regulator)
 Four the most common 81%.

 Mutation designated by ∆F508 is most common.
Occurrence almost 50% cases
PCR/OLA
 Like 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). 33
PCR/OLA
 Oligo 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.
34
PCR/OLA
35
PCR/OLA
 DNA 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.
36
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.
37
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.
38
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.
39
RAPD Analyses
RAPD
41
4.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
42
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.
43
Minisatellite DNA
 After 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.
45
Minisatellite 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.
46
DNA
Fingerprinting
47
Biological Reporter molecules
• Proteins that naturally fluoresce or luminesce, or that can be easily induced to
do so, may be used as biological reporters in a variety of ways.
• Genes encoding these bioreporter proteins may be used to engineer cells to
produce a measurable signal in response to a particular chemical or physical
agent in their environment.
• A gene for a fluorescent or luminescent protein is placed under the control of a
promoter that responds to certain environmental signals so that when the
promoter is activated, a fluorescent or luminescent signal is produced
1. Colored Fluorescent Proteins
2. Luciferase
3. Microbial Biosensors
Colored Fluorescent Proteins
Green fluorescent protein.
• The 238-amino-acid-long photoprotein green fluorescent protein, isolated from
the jellyfish Aequorea victoria, fluoresces green when it is exposed to
ultraviolet light.
• the incorporation of green fluorescent protein into cells allows intact living cells
to be monitored in real time.
Red fluorescent protein.
• A red fluorescent TETRAMERIC protein from Discosoma coral
• BY iterative cycle of random mutagenesis
• The production of monomeric red fluorescent protein required 33 mutations
• several drawbacks compared to the native tetrameric form of the protein,
including decreased brightness and reduced photostability.
LUCIFERASE
• The luciferase enzyme, which catalyzes a light-emitting reaction,
may be produced by a variety of different organisms, including
bacteria, algae, fungi, jellyfish, insects, shrimp, and squid.
• Luciferase genes from bacteria are termed lux genes
• In some applications, all five lux genes are utilized as a means of

monitoring the presence and concentrations of various compounds
in the environment, such as organic compounds, including phenol,
salicylate, benzene, trichloroethylene, ammonia, xylene, toluene,
and ethylbenezene, or metals, including cobalt, copper, iron, lead,
mercury, nickel, and zinc.
Bacterial Biosensors
 Bacterial 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 (luxCDABE) (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.
51
Bacterial Biosensors
 To 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.
52
Bacterial
Bisensor
53
Specialized for Different Purposes
 Reporter genes can be used to measure promoter
activity or tissue-specific expression.
Courtesy of Joachim Goedhart, Molecular Cytology, SILS, University
of Amsterdam.
Stowers Institute for Medical Research
Photo courtesy of Robb Krumlauf,
Figure 03.10A: (a) Since the discovery of

GFP, derivatives that fluoresce in
Figure 03.09: Expression of a lacZ gene can different colors have been engineered.
be followed in the mouse by staining for b-
galactosidase (in blue).

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Cytosine Adenine

In water, plants, soil and humans.

 Sensitivity: means that the test must be able to

 DNA Diagnostic Systems include:

 Genetic diseases often are due to mutations or

 These genes (DNA) can be used as diagnostic tools.

 This involves using a DNA probe during DNA

 Denaturation of both the probe and target.

 If there is sequence homology between the target

 DNA or RNA: long (>100 nts) or short (<50 nts): and

 Useful for detection of parasites

 PCR product now

 Detect following laser

 Mismatch gives no PCR

 Perfect match gives

 Sickle cell anemia is a genetic disease which is caused by a

 A (normal) glutamic acid and S (sickle) valine.

 In the homozygous state SS the red blood cells are irregularly

 Normal DNA sequence CCTGAGG (A)

 Mutant DNA sequence CCTGTGG (S)

 Two primers which flank the mutant region of the  globin

 The PCR products is digested with CvnI and separated by

 1400 different types mutation CFTR gene: (Cystic

 Four the most common 81%.

 The two probes are incubated with the PCR amplified

 For the mutant gene the nt at the 3’ end of probe x is a

 To determine if the mutant or wild type gene is

 Probe x is labeled at 5’ end with biotin

 Probe Y is labeled at 3’ end with digoxygenin.

 After washing a colourless substrate is added.

 If a coloured substrate appears this is indicative that

 RAPD is often used to show relatedness among DNA

 In this procedure arbitrary (random) primers are used

 A single primer is used which must anneal in 2 places on

 DNA fingerprinting analyses genotypic traits.

 DNA fingerprinting (DNA typing) is used to

In legal proceedings to identify suspects and clear

 Collection of sample e.g. hair, blood, semen, and skin.

 Examination of sample to determine if there is enough

 The variability is due to either a gain or lost of repeats

 An individual inherit one microsatellite from each

 The chance of finding two individuals within the same

 In other words an individuals DNA fingerprint is

• Luciferase genes from bacteria are termed lux genes

• In some applications, all five lux genes are utilized as a means of

 After a 15 min incubation the luminescence of the

 When the solution contained low to moderate levels of

 The procedure is rapid, simple, cheap and a good screen

Figure 03.10A: (a) Since the discovery of

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