This document discusses molecular diagnostics techniques. It begins by introducing molecular diagnostics and its significance in detecting pathogens, genetic mutations, and biomarkers. It then describes several key techniques used in molecular diagnostics, including nucleic acid amplification methods like PCR, isothermal amplification, and hybridization techniques. It also discusses methods like microarrays, genotyping, and mass spectrometry. The document provides examples of how these techniques are applied to detect various infectious diseases and genetic conditions.

1. Applications of Biotechnology In
Molecular Diagnostics
MODULE II
T. Y. BSC. BIOTECHNOLOGY

2. INTRODUCTION
• Molecular Diagnostics is the detection of pathogenic mutations in DNA and RNA samples to aid in
detection, diagnosis, subclassification, prognosis and monitoring response to therapy.
• It covers correct and accurate identification of causative agents like microbes in microbial diseases,
particular genetic sequences in genetic diseases and protein levels.
• Specificity and Sensitivity important tools in diagnosis.
• Nucleic-based diagnosis originate from localization, Identification, and Characterization of genes
responsible for human disease. It also determines how these genes and proteins are interacting in a cell.
It focuses upon patterns, gene and protein activity patterns in different types of cells.
• This diagnostics uncovers the sets of changes and captures this information as expression patterns. Also
called "molecular signatures," these expression patterns are improving the clinicians' ability to
diagnose diseases.

3. SIGNIFICANCE OF MOLECULAR DIAGNOSTICS
• Detection of infectious diseases such as human immunodeficiency virus (HIV), hepatitis B virus
(HBV), hepatitis C virus (HCV), human papilloma virus (HPV), cytomegalovirus (CMV), Chlamydia
trachomatis, Neisseria gonorrhoeae, and Mycobacterium tuberculosis.
• Other molecular tests are applied in forensic medicine, paternity testing, tissue typing, oncology, and
food and beverage testing.
• Molecular diagnostic testing can be performed on very small amounts of tissue obtained from
many different types of procedures, including small biopsies and fine-needle aspirates.
• Testing can even be performed on archival material (material that has been collected in the past
and fixed in formalin). It is usually not necessary to take extra tissue for these tests, since most of
them can be performed on tissue that is left over after routine diagnostic analysis.

4. TECHNIQUES INVOLVED IN MOLECULAR DIAGNOSTICS
• Nucleic Acid Amplification Techniques: PCR and its modifications, Isothermal amplifications
• Nucleic Acid Hybridization Techniques: FISH( Fluorescent In Situ Hybridization), Line probe
assay
• Microarrays
• Genotyping
• Mass Spectometry

5. NUCLEIC ACID AMPLIFICATION TECHNIQUES
1. PCR (Polymerase Chain Reaction)
• PCR is used in molecular diagnostics due to its exquisite sensitivity and specificity.
• PCR allows the amplification of millions of identical DNA copies from an originally small amount
of pathogen genome in a clinical sample.
Principle:
• The target DNA is first extracted then denatured at
high temperature.
• Specific oligonucleotide primers are annealed to the
DNA in a lower temperature, followed by an
extension phase during which the DNA polymerase
enzyme copies the template strand.
• This cycle is repeated, usually 30 to 40 times,
resulting in millions of identical DNA copies

6. Applications Of PCR:
• PCR is increasingly applied in the diagnosis of viruses, bacteria, parasites, and fungi.
• Qualitative PCR is widely used for a range of infections including herpesviruses (HSV, VZV, CMV,
EBV), enteroviruses, parvoviruses, respiratory viruses, SARSCoV, and poxviruses, as well as
general or specific bacterial screening, such as Neisseria meningitidis, Streptococcus pneumoniae,
Bordetella pertussis, and Borrelia spp
PCR Modifications used in Molecular Diagnostics
a. Real time PCR:
• Also known as qPCR or quantitative PCR
• The accumulation of amplification product is measured as the reaction progresses, in real time,
with product quantification after each cycle.
• Real-time PCR takes advantage of a fluorescent reporter, which gives signal in direct proportion
to the amount of PCR amplicon.
• The method allows real-time detection and quantification of PCR amplicons following each
thermal cycle.

7. • A melting curve analysis method
differentiates PCR amplicons according
to length and different guanine
cytosine contents.
• This can be used as an alternative to
sequencing for detection of the
different strains of the pathogen or
gene mutations related to antibiotic
resistance (e.g. meticillin*-resistant
Staphylococcus aureus and
vancomycin-resistant enterococci).
• Real-time PCR is rapid with high
sample throughput, and usually is
more sensitive and specific in
comparison to conventional methods.
Graph of qPCR

8. b. Ligase chain reaction (LCR):
• LCR is a modification of PCR. In this method,
two adjacent probes hybridize to one strand of
the target DNA. The small gap between the
two adjacent primers is recognized by a highly
specific thermostable DNA ligase, and ligated
to form a single probe. The ligated products
then serve as templates for the amplification
process. LCR allows the detection of only
single base pair mutations, and it is very
specific. The most common application of LCR
is in the diagnostics of Chlamydia trachomatis
in cervical and urine samples.
Ligase Chain Reaction

9. C. Multiplex PCR
• Multiplex PCR is a particular kind of reaction that
can amplify more than one locus linked to
multiple microbes, hence, helps in simultaneous
detection of various microorganisms by a single
PCR reaction. This method is just similar to the
previous method except that several specific
primers are combined in a single PCR assay.
• Several multiplex PCR assays have been reported in
which foodborne pathogens have been detected, for
instance, a rapid multiplex PCR simultaneously
detected five important pathogens including L.
monocytogenes, Shigella flexner, Salmonella
enteritidis, E. coli O157:H7, and S. aureus in
artificially contaminated pork. This highly specific,
efficient, and sensitive method also found that
80% meat samples were positive for these
pathogens by using five primer sets specific to these
microbes.

10. 2. Isothermal Amplification
• Unlike PCR, isothermal amplification techniques are based on a distinct enzyme that does not require
thermocycling (heating and cooling cycles), which can take several hours. Isothermal amplification is rapid; it
usually requires less than an hour to perform.
• However, the technique is currently fairly expensive. We describe here some of the best-known isothermal
amplification methods.
a. Nucleic acid sequence-based amplification
(NASBA):
• In NASBA analysis which is performed in isothermal
conditions, the target RNA is converted to double-
stranded DNA by using T7 RNA polymerase, RNaseH,
and a primer with a T7 promoter.
• The DNA acts as a template to produce multiple
copies of RNA with a polarity opposite that of the
target, which can be used for the production of
additional DNA templates.
• NASBA is also suitable for DNA, with slight
modifications in the early steps of the process.
• A recent example of NASBA technology in clinical
diagnostics is a real-time multiplex. NASBA assay for
the detection of Mycoplasma pneumoniae,
Chlamydophila pneumoniae, and Legionella spp. in
respiratory specimens

11. Transcription-mediated amplification (TMA): The principle of TMA is similar to NASBA, except
that it uses the RNaseH activity of reverse transcriptase, whereas NASBA uses a separate enzyme for
that. Due to the introduction of West Nile virus (WNV) in to the US since 1999, screening of primary
WNV infection in blood donors was initiated. Currently, TMA is referred to as the most sensitive
nucleic acid test commercially available for WNV infection, and is used on a large scale for blood
donor screening in the US. Another clinical example of a large-scale and worldwide use of TMA
technology is in the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine and
urogenital swab specimens.
Strand displacement amplification (SDA): SDA is another isothermal amplification method. The
first set of primers containing a restriction site is annealed to DNA template. Second primers are then
annealed adjacent to the first ones and start amplification, after which restriction enzyme HincII is
introduced in order to nick the synthesized DNA. An exonuclease deficient form of the Klenow
fragment of Escherichia coli DNA polymerase I starts the amplification again, displacing the newly
synthesized strands. Similar to other isothermal amplification techniques, the amplification process
of SDA is very efficient. SDA is used, for example, in the diagnosis of N. gonorrhoeae infection from
female endocervical swabs and male first-void urine samples.

12. Fluorescence in situ hybridization (FISH) :
• The FISH technique allows the detection of microbial
nucleic acid directly from the sample (or cultured
sample) without prior nucleic acid amplification.
• Briefly, the technique consists of specimen fixation on a
microscope slide, hybridizing the prepared sample
with a specific fluorescent-labelled probe, and visual
detection of the hybridization with a fluorescent
microscope.
• In medical microbiology, the technique is used, e.g. in
determination of antibiotic resistance and detection
of Helicobacter pylori from gastric biopsy specimens.
Identifying bacteria from CSF or Staphylococcus aureus
from blood cultures
NUCLEIC ACID HYBRIDIZATION TECHNIQUES

13. Line probe assay
• The line probe assay (LiPA) is another nucleic
acid hybridization test, in which specific
oligonucleotide probes are attached at known
locations on a nitrocellulose strip as parallel
lines and hybridized with biotin-labelled PCR
products.
• One of the widely used LiPA applications is
rapid detection of rifampicin resistance in
Mycobacterium tuberculosis.
• The LiPA technique is also applied in the
detection of antiviral drug-resistant
mutations of HBV.

14. Microarrays consist of a two-dimensional matrix of biomolecules which are printed or synthesized on a glass,
silicon, plastic or nylon membrane.
Microarrays can detect both nucleic acids and antibodies, which attach to the immobilized biomolecule, which can
be, e.g., an oligonucleotide or a protein. Positive reaction can be detected with highly advanced scanners by use of
target labelling with fluorescent probes or antibodies
MICROARRAYS

15. • Genotyping refers to the Identification of variations of different strains or subtypes of the pathogen at
nucleic acid level.
• Genotyping of PCR amplicons can be performed by direct sequencing, by reverse hybridization to
genotype-specific probes, by restriction fragment length polymorphism, or by detection of single
nucleotide polymorphisms with mass spectrometry.
• Direct sequencing is usually considered as the gold standard as it provides accurate and extensive data.
Genotyping plays an important role in the management of HCV infection, as response to treatment
considerably varies between different genotypes; patients with HCV genotype 2 or 3 infection respond
better to treatment than those with genotype 1 infection.
• Both direct sequencing and probe hybridization approaches are widely used for HCV genotyping.
• An example of genotyping currently moving into routine practice is human papilloma virus (HPV) typing.
Eight oncogenic HPV types (16, 18, 31, 33, 35, 45, 52, and 58) are responsible for 80% of cervical cancers
and cervical intraepithelial neoplasia worldwide; distinct patterns are seen in different regions.
Genotyping for the oncogenic HPV types enables risk assessment, personalized clinical management and
early intervention in women with mildly abnormal Pap results.
• Genotyping is also widely used for molecular epidemiology and in identifying antimicrobial resistance
and high-risk strains.
Genotyping

16. • Mass spectrometry allows the measurement of the molecular mass of a sample, which can be
utilized in various clinical diagnostic settings.
• In this technique, the sample is first ionized, the ions are separated according to their mass-to-charge
ratios, and finally the separated ions are detected according to the ion current. Ionization of the
sample can be done in various ways, of which matrix-assisted laser desorption/ionization (MALDI) is
widely used in the genotyping of single nucleotide polymorphisms.
• MALDI mass spectrometry is an important tool in typing of bacteria and viruses, which is useful,
e.g. in detecting hyper virulent or antimicrobial drug resistant strains. The technique has been
introduced, e.g. for genotyping of HCV and monitoring of quasi species (a cloud of variant RNA viruses
that arise from mutations over time within a viral isolate) in chronic HCV infection.
• It is also used in the detection of hyper virulent N. meningitidis strains; only five of the 13 serogroups
are frequently associated with invasive disease. Recently, a method based on broad-range RT-PCR
followed by electrospray ionization mass spectrometry has been applied for monitoring of global
spread of novel influenza virus genotypes.
Mass spectrometry

19. DIAGNOSTIC BIOMARKERS
• A Diagnostic Biomarker detects or confirms the presence of a disease or condition of interest,
or identifies an individual with a subtype of the disease.
• Biological markers (biomarkers) have been defined as “cellular, biochemical or molecular
alterations that are measurable in biological media such as human tissues, cells, or fluids.”
• Biomarkers are biological characteristics that can be objectively measured and evaluated as an
indicator of normal biological processes, pathogenic processes, or pharmacological responses to
a therapeutic intervention. In practice, biomarkers include tools and technologies that can aid in
understanding the prediction, cause, diagnosis, progression, regression, or outcome of
treatment of disease.
• Biomarkers can also reflect the entire spectrum of disease from the earliest manifestations
to the terminal stages.
• Biomarkers can also provide insight into disease progression, prognosis, and response to
therapy.

20. Capabilities of Biomarkers:
•Describing the events between exposure and disease
•Establishment of dose-response
•Identification of early events in the natural history
•Identification of mechanisms by which exposure and disease are related
•Reduction in misclassification of exposures or risk factors and disease
•Establishment of variability and effect modification
•Enhanced individual and group risk assessments

21. Examples Of Diagnostic Biomarkers
• Sweat chloride may be used as a diagnostic biomarker to confirm cystic fibrosis
•Certain cystic fibrosis transmembrane conductance regulator (CFTR) mutations may be used as diagnostic
biomarkers for evaluating treatment for cystic fibrosis, to select patients more likely to respond to particular
treatments (i.e., to serve as a predictive biomarker)
•Galactomannan may be used as a diagnostic biomarker to classify patients as having probable invasive
aspergillosis for enrollment into clinical trials of antifungal agents for treatment of invasive aspergillosis.
•Blood sugar or hemoglobin A1c (HbA1c) may be used as a diagnostic biomarker to identify patients with Type 2
diabetes mellitus (DM)
•Repeated blood pressure readings obtained outside the clinical setting in adults 18 years and older may be used as
a diagnostic biomarker to identify those with essential hypertension
•Glomerular filtration rate (GFR) may be used as a diagnostic biomarker to identify patients with chronic kidney
disease
•Ejection fraction may be used as a diagnostic biomarker in patients with heart failure to identify patients with a
subset of disease (those with low ejection fraction or preserved ejection fraction)

22. Role of markers in disease diagnosis
• Identification of individuals destined to become affected or who are in the ―preclinical
stages of the illness
•Reduction in disease heterogeneity in clinical trials or epidemiologic studies
•Reflection of the natural history of disease encompassing the phases of induction, latency and
detection
• Target for a clinical trial.
• Provide a means of monitoring the state of progression of disease and the effectiveness of
therapeutic options.

23. Immunodiagnostic Techniques
• Immunodiagnostic assays are procedures that utilize products of the immune response as integral
parts of the test. Basically, immunodiagnostic assays use antibodies generated either against a single
antigen or antigens associated with a specific analyte, pathogen, or disease condition.
•Immunodiagnostics Techniques Using:
1. Fluorogenic reporters
2. Electro-Chemiluminescent tags
3. DNA reporters
4. Label free immunoassays

24. Fluroscent Immunoassay using flurogenic reporters
•Fluorescent immunoassays were developed to detect patient antibodies to a large number of viruses, bacteria, and
parasites, and to detect auto antibodies in patients suffering from diseases.
•In the typical fluorescent antibody assay, blood specimens from patients suspected of having a pathogenic infection
were incubated with antigen preparations attached to glass microscope slides. Normally, these antigen preparations
consisted of whole formalin-fixed bacteria or parasites, or histological specimens cut to about 5 microns
thickness.
• If antibodies to the pathogen were present in the patient blood sample, they would specifically bind to the antigen
fixed on the slide. After incubating for 30 minutes to two hours, the patient antibody mixture was washed off and a
second antibody preparation, coupled with a fluorescent dye, was added. If patient antibodies were present, the
second antibody would specifically attach to them during the incubation period.
•The glass slides were then washed again, dried, and a mounting agent with counter stain was added to the slide
before cover slipping.
•Slides were then visually observed under a fluorescent microscope. Positive reactions were indicated by the antigen
preparation showing complete peripheral fluorescence, and strength of the reaction was determined by serial
titration of the blood specimen.

26. Examples Of Flurogenic Reporters
1. Fluorescein Isothiocyanate (FITC)
2. Tetramethylrhodamine Isothiocyanate (TRITC).
These isothiocyanates will crosslink to amino, sulfhydryl, imidazoyl, tyrosyl or carbonyl groups on a
protein.
However, only the derivatives of primary and secondary amines generally yield stable products. Reactions
are most efficient at pH 8-9, and must be performed in an amine-free buffer such as carbonate/bicarbonate.
3. DAPI (4', 6-diamidino-2-phenylindole) : DAPI is a fluorescent dye that binds selectively to double
stranded DNA and forms strongly fluorescent DNA-DAPI complexes with high specificity. It is commonly
used to detect mycoplasma in cell culture via fluorescence microscopy.
4. Propidium Iodide

27. Electrochemiluminescenc
e
•Electrochemiluminescent immunoassay (ECLIA) uses
electrochemical compounds that generate light electrochemically,
linked with the cycle reaction of the oxidative reduction.
•With optimization of reaction solutions, selection of appropriate
conjugation method, and development of suitable compounds such as
Ru(bpy)3
2+ and tetrapentylammonium (TPA), it became possible to
apply electrochemiluminescent technology to immunoassays
•Ru(bpy)3
2+ has a reaction site for the conjugation of analytes using
activation reagent such as NHS.
•The conjugate generates light on the surface of gold electrodes.
Ru(bpy)3 on solid phase and TPA are oxidized on the surface of
electrodes to form Ru(bpy)3
2+ and TPA+, respectively.
TPA+ spontaneously loses electrons.
•Ru(bpy)3
3+ can generate emission light at 620 nm when it returns to
Ru(bpy)3
2+ as a ground state through reduction with TPA.

28. •The efficiency of light generation (quantum yield) depends on the proximity between the electrode and the
conjugate and, thereby, on the diffusion mobility of the conjugate.
•Free conjugates can generate more light than conjugates fixed on the solid phase as a result of immune reaction.
This allows easy access to the homogeneous assay format, but relatively high background noise interferes with
assay sensitivity, as it does in other homogeneous assays.
•The assay protocol for the determination of analytes is as follows: magnetic microparticles coated with antibody
as the solid phase and sample mix together for immune reaction. After washing for bound and free conjugates by
magnetic separation according to the assay format, the solid-phase suspension is introduced into the detector
with the electrode to measure chemiluminescence.

29. DNA Reporters
• Genes that are used in genetic analysis because their products are easy to detect are known as
reporter genes. They are often used to report on gene expression, although they may also be
used for other purposes, such as detecting the location of a protein or the presence of a
particular segment of DNA , or even if two proteins interact.
•Easily assayble enzymes as DNA Reporters
1. ꞵ- Galactosidase
2. Alkaline Phosphatase
3. Luciferase
4. Green Fluorescent Proteins

30. 1. ꞵ- Galactosidase
•One of the first reporter genes for monitoring gene expression was the lacZ gene encoding β-galactosidase.
This enzyme normally splits lactose, a compound sugar found in milk, into the simpler sugars glucose and
galactose. However, β-galactosidase will also split a wide range of galactose compounds (i.e., galactosides) both
natural and artificial.
•The two most commonly used artificial galactosides are ONPG and X-gal. ONPG (o-nitrophenyl galactoside) is
split into o-nitrophenol and galactose. The o-nitrophenol is yellow and soluble, so it is easy to measure
quantitatively.
•X-gal (5-bromo-4-chloro-3-indolyl β-D-galactoside) is split into galactose plus the precursor to an indigo
type dye. Oxygen in the air converts the precursor to an insoluble blue dye that precipitates out at the location
where the lacZ gene is expressed.
Substrate 1: Lactose

31. Substrate 2: ONPG
Substrate 3:X -Gal

32. 2. Alkaline Phosphatase
Another reporter gene is the phoA gene that encodes alkaline phosphatase. This enzyme cleaves
phosphate groups from a broad range of substrates .
Like β-galactosidase, alkaline phosphatase will use a variety of artificial substrates:
(1) o-Nitrophenyl phosphate is split, releasing yellow o-nitrophenol.
(2) X-phos (5-bromo-4-chloro-3-indolyl phosphate) consists of an indigo dye precursor joined to
phosphate. After the enzyme splits this, exposure to air converts the dye precursor to a blue dye, as in
the case of X-gal.
(3) 4-Methylumbelliferyl phosphate releases a fluorescent compound when the phosphate is removed.
Substrate 1 Substrate 2

33. •Alkaline phosphatase removes phosphate groups from various substrates.
•When the phosphate group is removed from o-nitrophenyl phosphate, a yellow dye is released.
•When the phosphate is removed from X-phos, further reaction with oxygen produces an insoluble
blue dye as for Xgal.
•Additionally, alkaline phosphatase releases a fluorescent molecule when the phosphate is removed
from 4-methylumbelliferyl phosphate.

34. 3. Light Emission by Luciferase As a Reporter System
•A more sophisticated reporter gene encodes luciferase. This enzyme emits light when provided with a
substrate known as luciferin.
•Luciferase is found naturally in assorted luminous creatures from bacteria to deep-sea squid. The lux
genes from bacteria and the luc genes from fireflies produce different brands of luciferase, but both
work well as reporter genes.
FIG:
Luciferase Degrades Luciferin and Emits Light
Luciferase is an enzyme that alters the structure of
luciferin. When the structure is altered, a pulse of light
is emitted, which is detected by a photodetector.
The luciferin shown in this figure is FMN (flavin
mononucleotide), which is used by bacterial luciferases.

35. •The luciferins used by the different types of luciferase are chemically different.
•Bacterial luciferase uses the reduced form of the co-factor FMN (flavin mononucleotide) as its luciferin.
Oxygen and a long chain aldehyde (R-CHO) are also needed. Both the reduced FMN and the aldehyde are
oxidized.
•Different groups of eukaryotes make several chemically distinct luciferins that are used solely for light
emission.
•Firefly luciferase requires ATP as well as oxygen and firefly luciferin
•If DNA carrying a gene for luciferase is incorporated into a target cell, it will emit light only when the
appropriate luciferin is added. Although high-level expression of luciferase can be seen with the naked
eye, usually the amount of light is small and must be detected with a sensitive electronic apparatus such
as a luminometer or a scintillation counter.

36. Green Fluorescent Proteins as reporters
•Unlike the products of most reporter genes, green fluorescent protein (GFP) is not an enzyme, and it
does not need a nonprotein co-factor for it to fluoresce.
•GFP is a stable and nontoxic protein from jellyfish that can be visualized by its inherent green
fluorescence. Consequently, GFP can be directly observed in living tissue without the need for adding
any reagents.
•The cloned gene for GFP originally came from the jellyfish Aequorea victoria. This form of GFP is excited
by long wavelength UV light (excitation maximum 395 nm) and emits at 510 nm in the green. A variety of
genetically engineered variants of GFP are also in use.

37. 1. Fusions of regulatory regions and
promoters to the gfp gene have been used
to monitor the expression of many genes
(B) Phase contrast and (C) Fluorescence
emission of germlings of the fungus
Aspergillus nidulans. Original GFP was used
to label the nucleus and a red GFP variant
(DsRed) for the mitochondria
Applications of GFP

38. 2. GFP is widely used to localize proteins within the cell
GFP can be used to reveal where a protein is localized
within the cell. The first step is to fuse the GFP gene in
frame with all or part of the structural gene that encodes
the protein of interest. The fused construct is then
expressed in a host cell. The cells are excited with long
wavelength UV light and visualized under the
microscope. If the protein is normally located in the
membrane, as in this example, the cell membrane will
fluoresce green in the microscope.

39. Label Free Immunoassays
● Label-free technologies quantitate biomolecules by sensing a change in a physical parameter caused by
biomolecular interactions.
● Typically, the physical parameter is refractive index, optical thickness, energy, or mass.
● Using this approach, immunoassays based on label-free technologies, i.e. label-free immunoassays (LFIAs), are
able to measure antibody-antigen binding in real time, achieving immunometric quantitation without
attaching a reporter molecule (enzyme, fluorophore, etc.) to the immunocomplex.
● Conventional label-free technologies include surface plasmon resonance, isothermal titration
calorimetry, and quartz crystal microbalance.

40. Surface Plasmon Resonance
SPR-based biosensors are currently the most
commonly used devices for optical biosensing
applications. SPR-based sensing has recently
emerged as a valuable technique for measuring
binding constants, association and
dissociation rate constants, and
stoichiometry for bimolecular binding
interaction kinetics in a number of emerging
biological areas.
The practical applications of SPR analyses
includes kinetic analysis, equilibrium analysis
and concentration analysis. Kinetic and
equilibrium analyses are used to characterize
bimolecular interaction (ligand–analyte
binding, antibody–antigen interaction,
receptor characterization etc.) in a label-free,
real-time manner

41. Isothermal titration calorimetry
● Used for the label-free measurement of the binding affinity and thermodynamics of biomolecular
interactions to understand function and mechanisms at molecular level.
● Isothermal Titration Calorimetry is used to measure reactions between biomolecules.
● The methodology allows determination of the binding affinity, stoichiometry, and entropy and
enthalpy of the binding reaction in solution, without the need to use labels.
● When binding occurs, heat is either absorbed or released and this is measured by the sensitive calorimeter
during gradual titration of the ligand into the sample cell containing the biomolecule of interest.

42. Quartz Crystal Microbalance
● The quartz crystal microbalance (QCM) is a biosensor platform that incorporates a mechanical
transducer, which operates on the principle of mass detection.
● QCM-based biosensors have gained significant interest in the field of pathogen detection due to their
ability to detect virtually any type of biomolecule via a label-free method . Moreover, the rapid
detection process and the high sensitivity of QCM systems are particularly attractive for the development
of novel diagnostic tools.
● QCM systems can detect virtually any type of molecule because mass is an intrinsic property of all
substances, making it a versatile platform for detecting the diverse types of disease biomarkers.
● Molecular recognition events at the crystal surface are instantaneously reflected in the frequency
response without the need for labelling procedures, resulting in a short detection time, typically
between 30 min to 1 h.

43. Binding event increases the resolution of the frequency response, which effectively lowers the detection
limit of the biosensor.

44. Latest development in Label Free Immunoassays
● In the past decade, label-free technologies have advanced into the era of dip-in-solution sensing probes.
● The resulting LFIAs are open access, making them similar to plate-format assays without complicated sample
delivery or fluidics. This allows for simple experiment workflows and makes new LFIAs well-suited for clinical
laboratory applications.
● A new technology of this kind is thin-film interferometry (TFI), which incorporates a thin glass rod as a
sensing probe that transmits light to form thin-film interference on a sensing surface. When biomolecules
bind to the sensing surface and change its optical thickness, the interference pattern also changes relative
to the number of bound biomolecules. In this way, this method measures the quantity of bound
biomolecules in real time.

45. Cellular Genomics in Diagnostics

46. What is Cellular Genomics?
• Cellular genomics is the study of the genetic makeup of a single cell – from the cell’s entire
DNA code (its genome), to the secondary code that organises the genome (its epigenome), and
the total genetic output of the cell (its transcriptome). Exquisitely detailed and massively high-
throughput, cutting-edge cellular genomics technologies make it possible to unlock unknown
insights into how cells work individually, and how they function together.
• Cellular genomics can revolutionise our understanding of complex diseases, particularly
cancer, immunological and neurological diseases.

47. Overview of single-cell genomics to understand disease pathogenesis of heart failure

48. Single cell multi omics: The simultaneous detection of DNA sequences and RNA expression profiles
enables the identification of disease-causing variants and their association with gene expression.
There are novel methods to simultaneously extract information from DNA and RNA . By physically
separating mRNA from genomic DNA using oligo-dT bead capture and performing whole-transcriptome
and whole-genome amplifications.
Study of multiple perturbations with single cell readouts:Multiple perturbations with single-cell
readout has been developed to analyze epigenetic regulation , enhancer–promoter interactions, protein
expression , and morphological and phenotypical assessments.
Immunoprofiling: The diversity of the vertebrate adaptive immune system is based on somatic
rearrangements of V(D)J genes encoding the T-cell receptor (TCR) α and β chains; therefore, simultaneous
analysis of TCR sequence (clonality) and gene expression from individual cells provides a deeper
understanding of molecular behavior in the adaptive immune system. By integrating single-cell
transcriptomes with clonal information during the development of the human thymus.

49. • Human diseases often affect specific cell types within organ systems.
• In some disorders, such as amyloid lateral sclerosis (ALS), the disease impacts a single cell
type representing a minor population of all cells in the tissue, as is the case for the motor
neurons in ALS.
• Because tissues are composed of heterogeneous cell types, studying cell-type-specific features
of human pathological conditions demands alternatives to conventional genomics approaches,
such as bulk tissue RNA sequencing.
• Recent advances in single-cell genomics, in particular single-cell RNA sequencing, are
opening new avenues for identifying the exact molecular changes that are associated
with pathology in specific cell types

51. Functional Genomics in Diagnostics

52. What is functional genomics?
• Functional genomics is the study of how the genome and its products, including RNA and
proteins, function and interact to affect different biological processes.
• The field of functional genomics includes transcriptomics, proteomics, metabolomics and
epigenomics, as these all relate to controlling the genome leading to expression of particular
phenotypes.
• By studying whole genomes— clinical genomics, transcriptomes and epigenomes—functional
genomics allows the exploration of the diverse relationship between genotype and
phenotype, not only for humans as a species but also in individuals, allowing an
understanding and evaluation of how the functional genome ‘contributes’ to different
diseases.
• Functional variation in disease can help us better understand that disease.

53. Clinical Functional Genomic Pathway:
Functional genomic analysis can be performed on a patient sample using highthroughput technologies
like bulk RNA sequencing or newer methods, including spatial transcriptomics, to provide insight into
the cellular transcriptome. These are compatible with next generation sequencing (NGS) which is a
massively parallel, high-throughput technology that rapidly determines the order of nucleotides in entire
genomes or targeted regions .
It is also possible to use microarrays to profile multi-gene expression, however, while this method is
more cost-effective, it does not give the same complete picture as RNA sequencing or spatial
transcriptomics.
If only a small number of genes need to be tested, real-time PCR provides a highly sensitive and cost-
efficient method of choice.
Downstream bioinformatic analysis of the patient’s unique functional genome can identify diseases or
disorders, allowing for a tailored treatment plan specific to the patient. This more accurate diagnosis and
treatment results in better prognosis for patients and is the fundamental basis of precision medicine.

55. Use of functional genomics in diagnosing cancer
The field of functional genomics is of great relevance clinically to cancer patients, where
most research and development is currently focused. Changes in the genome or
epigenome can cause cancer by promoting uncontrolled cell growth or causing the
immune system to fail to destroy tumors.
Using a clinical functional genomics approach can allow for earlier and more accurate
cancer diagnosis, leading to more accurate treatment options and better prognosis for
patients.