INTRODUCTION TO MOLECULAR

DIAGNOSTICS
• Molecular diagnostics is a collection of techniques used to analyze
biological markers in the genome(in the fields of molecular biology
and genetics, a genome is all the genetic information of an organism)
and proteome(the entire set of proteins that is ,or can be expressed
by a genome , cell, or tissue, or organism at a certain time).
• By analyzing the specific of the patient and their disease, molecular
diagnostics offers the prospect of personalized medicine. This test are
useful in a range of medical specialities, including infectious disease,
oncology , human leucocyte antigen typing, coagulation and
pharmacogenetics.
Clinical diagnosis entering into new
phase
• Major advances have been made in the science of genetics ,resulting in the
increase use of molecular technology in clinical laboratory. A wide variety
of drugs in late preclinical and clinical development are being targeted to
disease specific gene and protein defects that will require co-approval of
diagnostics and therapeutics products by regulating agencies . To respond
to this demand, major pharmaceutical companies will form partnerships
with diagnostics companies or develop their own in-house capacities that
will permit efficient production of more effective and less toxic integrated
personalized medicine drug and therapeutics represents a major new
opportunity to emerge as leaders of the new medicine, guiding the
selection , dosage, route of administration and multidrug combination and
producing increased efficacy and toxicity of pharmaceutical products.
Application of molecular diagnostics
in the field of clinical diagnostics
Molecular diagnostic test
Molecular diagnostic focuses primarily on nucleic acids. Rapid advances in
molecular diagnostics both enable basic research and results in practical
diagnostic test. These type of tests include ;
. The analysis of DNA
. Analysis of RNA(nucleic acid)
.Micro RNA
.Complex proteomic or metabolomics pattern array based tests.
Molecular diagnostics technique
• Molecular diagnostics or DNA-based detection include a variety of
new and even experimental technologies, such as:
• Hybridization(probes), for example, clustered regularly interspersed
short palindromic repeats(CRISPR)-ca based assays
• Genotyping
• Sequencing, including nanopore sequencing
• Signal amplification
• Target amplification(polymerase chain reaction(PCR): singleplex and
multiplex.
Insitu hybridization
• Insitu hybridization(ISH) is a type of hybridization that uses a labelled
complementary DNA, RNA or modified nucleic acids strand (i.e probe)
to localize a specific DNA or RNA sequence in a portion or section of
tissue (in situ) or if the tissue is small enough(e.g plant seeds,
drosophila embryos) ,in the entire tissue (whole mount ISH), in
cells ,and in circulating tumor cells(CTCs). This is distinct from
immunohistochemistry, which usually localizes proteins in tissue
sections.
Insitu hybridization of wild type drosophila embryos
at different developmental stages for the RNA from
a gene hunchback.
Generation of riboprobes
Fluorescence in situ
hybridization(FISH)
• In situ hybridization is used to reveal the location of specific nucleic
acid sequences on chromosomes or in tissues , a crucial step for
understanding the organization, regulation and function of genes. The
key techniques in use include in situ hybridization to mRNA with
oligonucleotide and RNA probes (both radio-labelled and hapten-
labelled),analysis with light and RNAs and RNA plus protein, and
fluorescent in situ hybridization to detect chromosomal sequences.
Localization of a gene by fluorescence
in situ hybridization(FISH)
CHALLENGES OF IN SITU
HYBRIDIZATION
• In situ hybridization is a powerful technique for identifying specific
mRNA species within individual cells in tissue sections. Providing
insights into physiological processes and disease pathogenesis.
However, in situ hybridization requires that many steps be taken with
precise optimization for each tissue examined and for each probe use.
In other to preserve the target mRNA within tissues, it is often
required that cross linking fixatives (such as formaldehyde) be used.
• In addition, in situ hybridization on tissue sections require that tissue
slices be very thin. Common methods of preparing tissue sections for
insitu hybridization process include cutting specimens with a cryostat
or a compresstome tissue slicer.
Multiplex polymerase chain reaction
• Multiplex polymerase chain reaction refers to the use of polymerase
chain reaction to amplify several different DNA sequences
simultaneously( as if performing many separate PCR reactions all
together in one reaction ). This process amplifies DNA samples using
multiple primers and a temperature mediated DNA polymerase in a
thermal cycler. The primers design for all primers pairs has to be
optimized so that all primer pairs can work at the same annealing
temperature during PCR. Commercial multiplex kits for pcr are
available and used by many forensic laboratories to amplify degraded
DNA samples
Some of the amplication of
multiplex pcr include
• Pathogen identification
• High throughput analysis genotyping
• Mutation analysis
• Gene deletion analysis
• Template quantitation
• Linkage analysis
• RNA detection
• Forensic studies
• Diet analysis
Polymerase chain reaction
• Nucleic acid hybridization methods were mostly used methods and DNA
probes were the powerful tools in molecular diagnostics. although
hybridization methods are highly specific for detecting targets, they are limited
by their sensitivity. In 1985, saiki and his colleagues identify human
immunodeficiency virus(HIV) by using pcr method ,and this was the first
report of the application of pcr in clinical diagnosis of infectious disease. Since
then, pcr has become the method of choice for molecular diagnostic analysis
because
• (1) it is easy to perform
• (2) it has an open system that allows the user to easily design assays and
control reaction conditions
• (3) it satisfies clinical turn around times
Polymerase chain reaction
Masssive parallel sequencing
Massive parallel sequencing or massively parallel sequencing is any of
several high-throughput approaches to DNA sequencing using the concept of
massively parallel processing; it is also called next-generation sequencing (NGS)
or second-generation sequencing.
Template preparation methods for NGS
Two methods are used in preparing templates for NGS reactions: amplified
templates originating from single DNA molecules, and single DNA molecule
templates. For imaging systems which cannot detect single fluorescence
events, amplification of DNA templates is required. The three most common
amplification methods are emulsion PCR (emPCR), rolling circle and solid-
phase amplification. The final distribution of templates can be spatially random
or on a grid.
Emulsion PCR
• In emulsion PCR methods, a DNA library is first generated through
random fragmentation of genomic DNA. Single-stranded DNA
fragments (templates) are attached to the surface of beads with
adaptors or linkers, and one bead is attached to a single DNA
fragment from the DNA library. The surface of the beads contains
oligonucleotide probes with sequences that are complementary to
the adaptors binding the DNA fragments.
 The beads are then compartmentalized into water-oil emulsion droplets. In
the aqueous water-oil emulsion, each of the droplets capturing one bead is a
PCR microreactor that produces amplified copies of the single DNA template.
Gridded rolling circle nanoballs
• Amplification of a population of single DNA molecules by rolling circle
amplification in solution is followed by capture on a grid of spots sized to be
smaller than the DNAs to be immobilized.
• DNA colony generation (Bridge amplification)
• Forward and reverse primers are covalently attached at high-density to the
slide in a flow cell. The ratio of the primers to the template on the support
defines the surface density of the amp
Sequencing approaches

Pyrosequencing is a non-electrophoretic, bioluminescence method that

measures the release of inorganic pyrophosphate by proportionally
converting it into visible light using a series of enzymatic reactions.
Unlike other sequencing approaches that use modified nucleotides to
terminate DNA synthesis, the pyrosequencing method manipulates
DNA polymerase by the single addition of a dNTP in limiting amounts.
Upon incorporation of the complementary dNTP, DNA polymerase
extends the primer and pauses. DNA synthesis is reinitiated following
the addition of the next complementary dNTP in the dispensing cycle.
The order and intensity of the light peaks are recorded as flowgrams,
which reveal the underlying sequence.
•
Sequencing by reversible terminator chemistry
• This approach uses reversible terminator-bound dNTPs in a cyclic
method that comprises nucleotide incorporation, fluorescence
imaging and cleavage. A fluorescently-labeled terminator is imaged as
each dNTP is added and then cleaved to allow incorporation of the
next base. These nucleotides are chemically blocked such that each
incorporation is a unique event. An imaging step follows each base
incorporation step, then the blocked group is chemically removed to
prepare each strand for the next incorporation by DNA polymerase.
this series of steps continues for a specific number of cycles, as
determined by user-defined instrument settings. The 3' blocking groups
were originally c orceived as either enzymaticor chemical reversal The
chemical method has been the basis for the Solexa and Illumina
machines. Sequencing by reversible terminator chemistry can be a four-
colour cycle such as used by Illumina/Solexa, or a one-colour cycle such
as used by Helicos BioSciences. Helicos BioSciences used “virtual
Terminators”, which are unblocked terminators with a second
nucleoside analogue that acts as an inhibitor. These terminators have
the appropriate modifications for terminating or inhibiting groups so
that DNA synthesis is terminated after a single base addition.
Phospholinked Fluorescent Nucleotides or Real-time sequencing
• Pacific Biosciences is currently leading this method. The method of
real-time sequencing involves imaging the continuous incorporation
of dye-labelled nucleotides during DNA synthesis: single DNA
polymerase molecules are attached to the bottom surface of
individual zero-mode waveguide detectors (Zmw detectors) that can
obtain sequence information while phospholinked nucleotides are
being incorporated into the growing primer strand. Pacific Biosciences
uses a unique DNA polymerase which better incorporates
phospholinked nucleotides and enables the resequencing of closed
circular templates.
 Biosensor and
immunosensor based
diagnostics
A biosensor is an analytical device that integrates a biological element on a solid state surface,
enabling a reversible biospecific interaction with the analyte and a signal transducer. This
categories of sensors is based on antigen-antibody binding, nucleic acid hybridization and
receptor-ligand interaction.if antibodies or antibody fragments are applied as biological
element the device is called immunosensor. The immunosensors are affinity ligand-based
biosensing solid state device in which the immune-chemical reaction is coupled to a transducer.
• The fundamental basis of all immunosensors is the specificity of the
molecular recognition of antigens by antibodies to form a stable complex.
Immunosensors can be categorized based on detection principle applied.
The main developments are electro-chemical, optical, and micro
gravimetric immunosensors. There are four types of immunosensor
detection devices:
• (i) electrochemical (potentiometric, amperometric
or,conductometric/capacitative);
• (ii) optical;
• (iii) microgravimetric and
• (iv) thermometric
Principle of biosensor
• There are two different types of biosensors: biocatalytic
andbioaffinity-based biosensors. The biocatalytic biosensor uses
mainly enzymes as the biologicalcompound, catalyzing a signaling
biochemical reaction. The bioaffinity-based biosensor, designedto
monitor the binding event itself, uses specific binding proteins,
lectins, receptors, nucleic acids,membranes, whole cells, antibodies
or antibody related substances for biomolecular recognition.In
affinity sensors, the recognition between the analyte in solution and
the immobilized biologicalelement is based on an affinity reaction.
Biosensors have been developed to detect hybridization ofunlabelled
DNA using different transduction methodologies.
 Electrochemical detection: The great advantage of the DNA electrochemical
biosensor isthat it does not require very expensive components. These
are based on the voltametricmonitoring of DNA hybridization. Voltametric
responses of these indicators are influencedby the result of DNA
hybridization. Some electroactive DNA intercalators and minor groovebinding
substances as redox indicators of the hybridization events are available. A
secondapproach directed to hybridization detection is based on the
electroactivity of nucleic acids.Single-stranded and duplex DNA can easily be
recognized as the basis of the intrinsic DNAelectroactivity (adenosine and
guanine) without any redox indicator. Signal of single-stranded DNA
obtained at the electrodes differ from those produced by double-
strandedDNA, thus making it possible
DNA Microarray
• DNA microarray is a collection of microscopic DNA spots attached to a
solid surface. Scientists use DNA microarrays to measure the expression
levels of large numbers of genes simultaneously or to genotype multiple
regions of a genome. Each DNA spot contains picomoles (10−12 moles)
of a specific DNA sequence, known as probes (or reporters or oligos).
These can be a short section of a gene or other DNA element that are
used to hybridize a cDNA or cRNA (also called anti-sense RNA) sample
(called target) under high-stringency conditions. Probe-target
hybridization is usually detected and quantified by detection of
fluorophore-, silver-, or chemiluminescence-labeled targets to determine
relative abundance of nucleic acid sequences in the target.
DNA microarray
Hybridization of the target to
the probe
• The core principle behind microarrays is hybridization between two DNA
strands, the property of complementary nucleic acid sequences to
specifically pair with each other by forming hydrogen bonds between
complementary nucleotide base pairs. A high number of complementary
base pairs in a nucleotide sequence means tighter non-covalent bonding
between the two strands. After washing off non-specific bonding
sequences, only strongly paired strands will remain hybridized.
Fluorescently labeled target sequences that bind to a probe sequence
generate a signal that depends on the hybridization conditions (such as
temperature), and washing after hybridization. Total strength of the signal,
from a spot (feature), depends upon the amount of target sample binding
to the probes present on that spot.
Uses and types

Many types of arrays exist and the broadest distinction is whether they are spatially arranged on a
surface or on coded beads:

The traditional solid-phase array is a collection of orderly microscopic "spots", called features,
each with thousands of identical and specific probes attached to a solid surface, such as glass,
plastic or silicon biochip (commonly known as a genome chip, DNA chip or gene array). Thousands
of these features can be placed in known locations on a single DNA microarray.
The alternative bead array is a collection of microscopic polystyrene beads, each with a specific
probe and a ratio of two or more dyes, which do not interfere with the fluorescent dyes used on
the target sequence.
• DNA microarrays can be used to detect DNA (as in comparative genomic hybridization), or
detect RNA (most commonly as cDNA after reverse transcription) that may or may not be
translated into proteins. The process of measuring gene expression via cDNA is called expression
analysis or expression profiling.
• Antibody Based Diagnostics: Serological Reactions (Serology)Antigen-
antibody interactions in vitro (under laboratory conditions) are referred to
as serologicalreactions or serology (so named because they commonly
involve patients serum) .Antigen-antibody interactions were first
adapted to laboratory tests to diagnose disease in the late1800s. At
present, they have become a highly sophisticated and often automated
discipline ofimmunology, and are widely used in clinical diagnosis,
epidemiology, and basic research. A varietyof serological reactions are
used in laboratories, the most important and often used ones
are:precipitation-test, agglutination-test, fluorescent-antibody technique
(immunofluorescence),readioimmunoassay (RIA), and enzyme-linked
immunosorbent assay (ELISA).
• Precipitation-test (Precipitin-Test)
• When soluble antigens and the homologous antibody molecules react, they sometimes
form largepolymeric macromolecules terminating into visible precipitate . The
precipitationoccurs in two stages: first, the antibodies bind to antigens forming antigen-
antibody-complex withina few seconds or minutes, then the constant regions of antibodies
of the complexes bind to each390 Biotechnology in Medicine and Agricultureother within
some hours resulting in the formation of visible precipitate. The formation of precipi-tate is
dependent on the proper relative concentrations of the antigen and antibody molecules in
aspecific region called the zone of equivalence or zone of precipitating, i.e., the zone of
equivalence(or precipitation) defines the region wherein theconcentrations of antigen and
antibody moleculesreach almost equivalence. Precipitation tests are performed either in
fluid-fluid precipitation, or gel-gel precipitation. In the former, the solutions of antigen and
antibody are layered over each otherin a thin tube, whereas the diffusion of antigen and
antibody takes place through a semisolid gel(such as agarose).
• . In the latter, mainly applied in the laboratory diagnosis of bacterial
infections ofhumans and important animals, the precipitation tests
are also used for many other purposes suchas
• (i) identification of blood or seminal fluid in stains on clothing,
weapons, etc.;
• (ii) postmortemdiagnosis of anthrax from a dead or decomposed
animal;
• (iii) detection of food adulteration and
• (iv)determination of the kind of an animal a mosquito has recently
fed on, to help preventing spreadof arthropod-borne diseases.
(a) Antigen-antibody interactions (b) precipitation-test
• Agglutination-testIn agglutination-test visible clumping or aggregation of cells or
particles takes place due to thereaction of surface-bound antigens of such cells or
particles with homologous antibodies . Pathogens causing many diseases like
typhoid fever (Salmonella typhi), gonorrhoea(Neisseria gonorrhoeae), rickettsial
diseases are detected by agglutination-tests; it is probablybest known for its
use in human red blood cells (RBCs) possess either type A or type
Bpolysaccharide antigens, or both type A and type B polysaccharide antigens or
neither of thesetwo antigens on their surface respectively. Blood types are
determined by mixing known antisera(anti-A and anti-B antibodies) with a blood
possesses type A but not type B antigens on thesurface of RBCs; type B blood
has the type B but not type A antigens on the surface of RBCs;type AB blood
possesses both type A and type B antigens on the surface of RBCs; and typeO
blood has neither type A nor type B antigens on the surface of RBCs.
• Agglutination-test (1 = cells or particles with antigens on their
surface, 2 = homolo-gous antibodies, 3 = clumping or agglutination
of cells or particles).
• In those cases where the antigens are not present or cell on particle surfaces and
remain freein soluble-state, the direct agglutination-tests normally fail. For
detection of such antigens,passive agglutinization-test is employed. In passive
agglutinization-test, the soluble antigens aretaken out, are first attached to the
surface of one of the carriers like latex beads, polystyrene,particle, red blood
cells, and then mixed with patients serum. Homologous antibodies presentin
serum attach with antigens present on the surface on the surface of the
carrier formingantigen-antibody-complexes that agglutinate. An excellent
example of passive agglutination-testusing latex beads as carrier is one of the
modern pregnancy test; other example is the detectionof pathogens like
Haemophilus influenzae (Haemophilus meningitis), Streptococcuspneumoniae
(pneumonia), Neisseria meningitides (meningococcal meningitis),
Treponemapallidum (Syphilis), Rubella virus (German measles).
• Fluorescent-antibody technique (immunofluorescence)The
fluorescent-antibody technique (immunofluorescence) is often used
to identify unknownantigens. The technique is based on the
behaviour of certain dyes, which fluoresce (glow) whenexposed to
certain wavelengths of light. Such dyes are: fluorescenin
isothiocyanate, which emitsan apple-green glow, and rhodamine
isothiocyanate, which emits orange-red light. Fluorescent-antibody
technique may be direct or indirect. In direct method, known
antibody molecules areconjugated (labelled) with a fluorescent dye;
the dye-labelled antibodies combine with thosemicrobes that
possess complementary antigens on their surface.
• The mixed microbial populationis viewed under fluorescence microscope
with an excitation wavelength appropriate for the dye;only dye-labelled
antibody attached microbial cells fluoresce and become visible. In
indirectmethod, the initially applied antibody is not labeled (conjugated)
with the dye. Instead, a secondlabeled antibody is applied which binds
the fluorescence label to the specific antibody that hasalready reacted
with its complementary antigen present on the surface of the microbial
cells inthe mixed population. Fluorescent-antibody technique
particularly helps identifying specificstrains of microorganisms within a
mixed microbial population; it is also useful in identifying
thosepathogenic microbes that are difficult or impossible to culture in
vitro.
• Radioimmunoassay (RIA)
• Radioimmunoassay (RIA) is a widely accepted and highly sensitive serological test in which one
of the reactants antibody, antigen or hapten is radio labelled with radioactive isotopes of
elements like iodine (125I) or hydrogen (3H) are detected in situ by radio autography . This
technique was developed in 1960s to detect hormones such as insulin, and is now used to
quantify very low levels of polypeptides, steroids thyroid hormones, vitamin B12, and viruses.
The steps involved in radioimmunoassay are:
• (i) A sample containing an unknown quantity of antigenic substance and its specific
antibodies, that react to form antigen-antibody-complex, is taken
• (ii) radio labelled antigenic substance is added which combines with unreacted antibody
molecules in the sample forming radio labelled antigen-antibody complex;
• (iii) the radio-labelled antigen-antibody-complex is separated from the sample and its amount is
determined ,and
• (iv) the concentration of the unknown antigenic substance in the sample calculated.
• The basis of radioimmunoassay is the competition between a known
amount an antigenic substance that is radio labelled and an
unknown amount of the same antigenic substance that is non-
radio labelled for the same antibody. The relative amounts of radio
labelled and non-radio labeled antigenic substance that bind with the
antibody molecules indicated the levels of the antigenic substance in
the sample. High levels of antigen-antibody-complex (radio labelled)
indicate a low level of unknown antigenic substance whereas low
level of radio labelled antigen-antibody-complex indicates a high
level of unknown antigenic substance in the sample
Enzyme-linked immunosorbant assay

• ELISA was pioneered by two groups of scientists, one in Sweden by Engvall

and Perlmann, andthe other in Holland by Van. Weeman and Schurs in
1972, and was developed by Adams and Clarkin 1977. ELISA is based on,
as the name suggests, enzyme-linked antibodies adsorbed on somesolid
surface . The most commonly employed enzyme is alkaline phosphatase
(otherenzymes used are peroxidase, glucose oxidase, p-galactosidase,
malate dehydrogenase, etc.) andthe solid surface is that of micro-ELISA
plates having shallow walls (depressions, capacity 0.4 ml)and made up of
polystyrene which has the property to bind with antigen or antibody
covalently.There are two methods of ELISA: indirect-ELISA used for the
detection and measurement ofantibody and direct-ELISA used for the
detection of antigen.
• ELISA is advantageous over other methods of serology because of its
simplicity, less expensive-ness, sensitivity, and accuracy similar to that
of radioimmunoassay (antigens and antibodies detect-able at levels of
about 10-10 g/ml or part in ten billion), stability of reagents (reagents
remain fullyfunctional both immunologically and enzymatically
throughout the process), and most importantly,the lack of radiation
hazards as the radioisotopes are not used. ELISA, however, is a time
savingdevice and can be completed within hours even in
laboratories with rudimentary facilities if prepared enzyme-
conjugatedantibodies are available.
Mass spectrometery

Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio
of ions. The results are presented as a mass spectrum, a plot of intensity as a function of the mass-
to-charge ratio. Mass spectrometry is used in many different fields and is applied to pure samples
as well as complex mixtures.

A mass spectrum is a type of plot of the ion signal as a function of the mass-to-charge ratio. These
spectra are used to determine the elemental or isotopic signature of a sample, the masses of
particles and of molecules, and to elucidate the chemical identity or structure of molecules and
other chemical compounds.

• In a typical MS procedure, a sample, which may be solid, liquid, or gaseous, is ionized, for
example by bombarding it with a beam of electrons. This may cause some of the sample's
molecules to break up into positively charged fragments or simply become positively charged
without fragmenting.
Part of a mass spectrometery

A mass spectrometer consists of three components: an ion source, a mass

analyzer, and a detector. The ionizer converts a portion of the sample into
ions. There is a wide variety of ionization techniques, depending on the
phase (solid, liquid, gas) of the sample and the efficiency of various
ionization mechanisms for the unknown species. An extraction system
removes ions from the sample, which are then targeted through the mass
analyzer and into the detector. The differences in masses of the fragments
allows the mass analyzer to sort the ions by their mass-to-charge ratio.
The detector measures the value of an indicator quantity and thus
provides data for calculating the abundances of each ion present. Some
detectors also give spatial information, e.g., a multichannel plate.
Basic Principle
A mass spectrometer generates multiple ions from the sample under investigation, it then separates
them according to their specific mass-to-charge ratio (m/z), and then records the relative abundance
of each ion type.

• The first step in the mass spectrometric analysis of compounds is the production of gas phase ions
of the compound, basically by electron ionization. This molecular ion undergoes fragmentation.
Each primary product ion derived from the molecular ion, in turn, undergoes fragmentation, and so
on. The ions are separated in the mass spectrometer according to their mass-to-charge ratio, and
are detected in proportion to their abundance. A mass spectrum of the molecule is thus produced.
It displays the result in the form of a plot of ion abundance versus mass-to-charge ratio. Ions
provide information concerning the nature and the structure of their precursor molecule. In the
spectrum of a pure compound, the molecular ion, if present, appears at the highest value of m/z
(followed by ions containing heavier isotopes) and gives the molecular mass of the compound.
• Components of a mass spectrometery
With all the above components, a mass spectrometer should always perform the
following processes:
Produce ions from the sample in the ionization source.
Separate these ions according to their mass-to-charge ratio in the mass analyzer.
Eventually, fragment the selected ions and analyze the fragments in a second
analyzer.
Detect the ions emerging from the last analyzer and measure their abundance
with the detector that converts the ions into electrical signals.
• Process the signals from the detector that are transmitted to the computer
and control the instrument using feedback.
 The impact of molecular approaches
to infectious disease diagnostics
There are many different approaches to the design of molecular diagnostic
tests. Pathogen detection can be achieved through a number of different
methods, including real-time polymerase chain reaction (PCR) detection
with melt-curve analysis, fluorescent in situ hybridization (FISH)-based
detection, and microarray-based detection, to name a few. Some tests
provide full automation of all necessary detection steps from sample receipt
to diagnostic result (sample-to-result), while others require offline steps
such as nucleic acid extraction and amplification. The results of a molecular
test can be qualitative or quantitative in nature.
• By targeting the genomic material unique to each pathogen,
molecular tests can deliver diagnostic results much quicker and, in
many cases, more accurately than conventional culture-based
techniques.
Application of molecular Diagnostics
in cancer
• Molecular diagnostics can be used to determine whether a person is
at risk for a certain type of cancer. When used this way, the tests may
also be referred to as molecular profiling or molecular risk
assessment. These tests help a person determine how likely he or she
is to develop cancer.
Testing for gene expression by older methods only
allows monitoring of a few genes at a time. GeneChip
analysis allows researcher to monitor hundreds to
thousands of genes simultaneously. By monitoring
many genes at once, sets of genes can be recognized
as having altered expression. GeneChip analysis
employs a small quartz chip to which known
fragments of DNA are attached. The DNA fragments
on the chip may represent all of the genes in a cell.
• Congenital heart disease (CHD) is a structural abnormality of the
heart and/or great vessels that is present at birth. CHD is the most
common birth defect with a reported prevalence of approximately 1%
of all live births.
• The rapid advancements in DNA sequencing technology, bioinformatics,
and computing infrastructure for genomic data processing, have led to a
shift in CHD genetic diagnosis from single loci testing to high throughput
genome-wide testing. Traditional genetic analytical techniques, including
karyotyping, Sanger sequencing, fluorescence in situ hybridization
(FISH), and multiple ligation dependent probe amplification still play
important, and sometimes indispensable, roles in clinical diagnostic
workups of patients with CHD. However, high throughput genomic
testing including chromosomal microarray (CMA), whole exome
sequencing (WES), and whole genome sequencing (WGS) are
increasingly used in clinical practice for patients with CHD.
Role of Molecular Testing for Disorders of Hemostasis
As with all diagnostic algorithms, determination of the cause of inherited bleeding disorders
begins with the patient history. The initiation of any laboratory investigations, be they phenotypic
or genetic in nature, should always be adequately supported by the patient’s personal and/or
family history of a significant hemostatic problem.
• For bleeding disorders, the recent emergence of interest in a variety of bleeding assessment
tools has provided a more quantitative clinical evaluation than previously available through non-
standardized history taking . The adoption of a standardized and quantitative bleeding
assessment has a number of pragmatic advantages including the enhancement of
communication of the significance of bleeding between health care professionals and,
importantly for the extent of subsequent laboratory evaluation, a more objective determination
of bleeding severity. Nevertheless, in young children and males, in whom the opportunities for
bleeding events are less evident, false negative bleeding scores may still be a challenge. In these
instances, the presence of a family history of excessive bleeding may still be an important trigger
for the initiation of laboratory investigation
Other Diagnostics method
In Vitro Diagnostics
Molecular diagnostics are often referred to as in vitro diagnostics. The words in vitro are
Latin
for “in glass” and refer to the glass test tubes in which the tests were originally performed.
Today, the phrase in vitro diagnostics refers to tests that are conducted on samples taken
from the body, such as blood, saliva, or cells from a tumor. In vitro diagnostics can be
contrasted with in vivo diagnostics such as ultrasound, X-rays, and computed tomography
(CT) scans, which are performed on a living person and produce an image. Today, all
molecular diagnostics are performed in vitro, and sometimes the whole field of molecular
• diagnostics is referred to as in vitro diagnostics_x0000_
Tissue Sampling
The first step in molecular diagnostics is to obtain tissue or specimen for testing. Tissue
samples are collected by different methods depending on the purpose and the type of test.
Blood samples are often drawn from veins in the arm. Urine can be studied, as can saliva
obtained from the mouth. Samples may be taken from the skin following a local anesthetic.
When samples need to be obtained from a solid tissue abnormality or tumor, the simplest
and least invasive option is a fine needle biopsy, in which a fine needle is inserted into the
• tissue and cells are aspirated._x0000_
• If a larger amount of tissue is needed, a core needle biopsy may be
used to remove cells and a small amount of surrounding tissue.
Surgical procedures may also be used when the removal of an even
larger amount of tissue is needed: an incisional biopsy removes a
portion of the abnormality and an excisional biopsy removes the
entire abnormality or tumor. Cells can also be obtained by scraping
tissues that naturally open to the environment, such as the cheek and
cervix. Another method involves the use of a flexible, lighted
instrument called an endoscope that is inserted into one of the body’s
natural openings. The endoscope allows the physician to see abnormal
areas on the lining of organs and pinch off tiny bits of tissue._x0000_
Importance of molecular Diagnostics
Why Molecular Diagnostics Are Important
• The medical community has recognized the importance of molecular
diagnostics for several decades, and this field is especially important
to cancer care. Molecular diagnostics have already improved cancer
diagnosis and treatment techniques, and research is continuing.
The image above shows the many areas in which molecular diagnostics
could be used. This spans the entire scope of cancer care beginning
with risk assessment and moving through to surveillance after diagnosis
and treatment.
• Before the use of molecular diagnostics, clinicians categorized cancer
cells according to their pathology, that is, according to their
appearance under a microscope. Borrowing tools from two new
disciplines, genomics and proteomics, molecular diagnostics
categorizes cancer-using technology such as mass spectrometry and
gene chips.
• Molecular diagnostics analyzes how these genes and proteins are
interacting inside a cell. The focus is on patterns--gene and protein
activity patterns--in different types of cancerous or precancerous
cells. Molecular diagnostics uncovers these sets of changes and
captures this information as expression patterns. Also called
"molecular signatures," these expression patterns are improving the
clinicians' ability to diagnose cancer and to recommend more
targeted therapies based on an individuals molecular signature.
What is the role of gene analysis in molecular diagnostics?
DNA microarrays, sometimes called "gene chips," allow researchers to
see the expression of hundreds or thousands of genes at one time. A
DNA microarray is a thin-sized chip with thousands of single-stranded
DNA fragments corresponding to various genes of interest that have
been inserted into "spots" in the microarray.
• A single microarray may contain 10,000 or more spots with each spot
containing pieces of DNA from a different gene. A single gene chip can
even hold representative fragments from the entire human genome.
• How arrays are use
What is the role of protein analysis in molecular diagnostics?
• Molecular diagnostics evaluates proteins in a cell, tissue, or organism,
including the shape, function, and patterns of expression. Research
seeks to identify proteins involved when normal cellular pathways
support malignant growth. In cancerous tissue, some of the proteins
critical for normal communication are damaged, inactive, overactive,
or missing entirely. The full set of proteins disrupting cellular
communications may vary from one cancer type to another, and they
may also vary somewhat from one patient to another with that type
of cancer.
• Molecular Diagnostics in oncology
Disadvantages of Molecular Testing
Molecular methodologies, while highly advantageous, do contain limitations
and certain disadvantages. These can include:
Cost: Molecular methodologies are usually more expensive than standard
traditional methodologies. Equipment and reagent costs could be prohibitive
to some laboratories. As molecular methods become more standard, the costs
could potentially decrease. Currently, laboratories that consider the cost
prohibitive prefer to transport molecular specimens to a reference laboratory.
Personnel requirements: Depending on laboratory accreditation
requirements and testing methodologies some personnel may not be
qualified to competently perform molecular testing.

• Laboratory space requirements: Molecular amplification methods

require dedicated space that may not be available in some clinical
laboratories.
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