MOLECULAR

DIAGNOSTICS
OF NON-
INFECTIOUS
DISEASES
dr. Siti Nur Aisyah Jauharoh, PhD
Introduction

Molecular diagnosis of human disorders is referred to as the

detection of the various pathogenic mutations in DNA and /or
RNA samples in order to facilitate detection, diagnosis, sub-
classification, prognosis, and monitoring response to therapy.
History of Molecular Diagnostics
3

The Molecular Biology Timeline

1865 Gregor Mendel, Law of Heredity

1866 Johann Miescher, Purification of DNA
1949 Sickle Cell Anemia Mutation
1953 Watson and Crick, Structure of DNA

1970 Recombinant DNA Technology

1977 DNA sequencing
1985 In Vitro Amplification of DNA (PCR)
2001 The Human Genome Project
 4

Impact on Human Diseases:

Practical application
 Diagnostic-Identity of a disease
 Prognostic-Outcome of a disease
 Predictive-Possibility of a disease
 Therapeutic-Response of a disease
to treatment
 5

Impact on Human Diseases

INFECTIOUS
DISEASE
HEMATOLOGY

Molecular
Pathology

SOLID
IDENTITY TUMORS
TESTING

GENETIC
DISEASE
 6

Molecular Diagnostics are Transforming Medicine

Recurrence monitoring
Molecular
diagnostics is
>$3 billion Drug selection
market WW and
growing at >20%
annually
Disease detection

Disease predisposition

Pre-natal testing Key questions

“Is the baby “What diseases “Has this “What drugs “How has the
healthy? “ is this patient at patient a should I disease
risk for?” disease?” prescribe?” returned?”

-> Need for Molecular tests

 7

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.
 8

Molecular Diagnostics in Multiple Disease Areas

Genetic • High throughput testing for genetic disorders
Testing including single nucleotide polymorphisms
(SNPs) markers, insertions, deletions
• Examples: Factor II, Factor V

Prenatal • Non-invasive detection of fetal diseases • Progress is being made

Diagnostics • Examples: Down syndrome, cystic fibrosis in all of these areas

• Each of these areas are

commercially attractive

Oncology • Early diagnosis of cancer • In some cases, the

• Example: circulating tumor DNA MassARRAY platform is
uniquely qualified for
specific tests

Transplantation • Non-invasive, early detection of organ • More tests will be added

Medicine rejection to the platform as these
• Example: urine testing for kidney rejection tests are rolled out

• Pathogen identification and early detection

Infectious • Examples: identification of multi drug resistant
Disease mycobacteria, early detection of drug-resistant
viral strains, e.g. HIV, HBV, HCV
 9

Pre-natal Diagnostics
130 million live births worldwide per year
 8 million live births in US and Europe per No accurate,
year non-invasive
prenatal
 6% of all babies are born with birth defects diagnostic
 over 900 fetal genetic disorders
tests (“NIPD”)
Down syndrome is the most common chromosomal
available
abnormality
 Although risk increases with age, 80% of Down
births are in women <35 years old

-> at the moment invasive methods available ->

require a certain amount of fetal cells e.g.
test for Down syndrome (Amniocentesis)
-> only non-invasive method: Ultrasound scanning
-> good to have a fast test for genetic disorders
like Hemoglobinopathies, Cystic fibrosis, Down
Syndrome
 10

Genetic Testing
Types of Mutations Tested
Few recurrent
mutations?
Point mutations?
Many unique
mutations?

Disease

Whole gene?
Some exons?
Deletions &
duplications?
Other mutations? Also with point
(Chromosomal mutations?
rearrangements)
 DETEKSI ANTIGEN MOLEKULAR
1. PCR based diagnostic
Antigenics : viruses, parasites, etc

a. PCR (Polymerase Chain Reaction)

• Desain Primer PCR & Optimasi Reaksi

• Protokol Standard PCR
• Deteksi amplicon (spectrofotometri &
electrophoresis)
b. Reverse Trancription PCR – RT PCR

Reaction : mRNA

cDNA

Protein
2. DNA SEQUENCING

• Aplication : Paternity testing, forensic science,

population studies, evolutionary,
gene therapy etc.
• Methode : Sanger et al
• How to Sequence DNA ?
Know the sequence
5’ GTGCACCTGACTCCTGTGGAG
CACGTGGACTGAGGACACCTC
2. DNA SEQUENCING

Select a primer
Single 5” primer

CACGTGGACTGAGGACACCTC 3’
Setting up cycle sequencing :
Synthesize from 5’ to 3’ using a single 5’ primer 4 different cycle seq.
reaction :
GTGCACCTGACTC
GTGCACCTGACT
GTGCACCTGAC
GTGCACCTGA
GTGCACCTG
GTGCACCT
GTGCACC
GTGCAC
GTGCA
GTGC The A lane will show
2. DNA SEQUENCING

ddATP ddCTP ddGTP ddTTP

Detection of Electrophoresis : GTGCACCTGACTA…

 DNA Sequencing
dideoxy method (Sanger)
Electrophoresis detection for DNA sequencing
4. RFLP Restriction Fragment Length Polimorphism

• Restriction Enzyme : cutting the specific

sites of DNA fragment.
• Aplication : Paternity testing, forensic sci.,
population studies, evolutionary, gene therapy etc.
• Recognition sites : 4-6 bases
• mutation sequences : unrecognize site
• Blunt & Stiky end
• How to choice the specific restriction
enzym ?
1. Mapping whole restriction enzymes Computerised on
sequnce DNA by Programe of GCGMap, MapPlot,
MapSort, PlasmidMap
2. Sequences mapping by SeqLab programe
3. Specific of Restriction enzymes.

e.g. Eco R1

E = genus Escherichia
co = species coli
R = Strain RY 13
I = First RE isolated from this species
 MOLECULAR ANTIBODY
DETECTION

1. MONOCLONAL & POLYCLONAL

ANTIBODY

2. Ig G, Ig M, Ig A
II. ANTIBODI MONOKLONAL (mAb)

Diproduksi dari sel hibridoma : fusi antara 2 sel yang

mempunyai galur genetika berbeda.

Hibridoma alamiah : zygot, fusi antara 2 sel kelamin yang

mempunyai galur genetik berbeda.

Hibridoma hasil rekayasa :

fusi antara sel plasma dan sel myeloma 
menghasilkan antibodi monoklonal (mAb).
Cara Produksi mAb

1. Mencit diimunisasi dengan antigen spesifik.

2. Keluarkan limfosit/sel B dari lien.
3. Biakkan dan fusikan dengan “myeloma cell
line” dengan memberi PEG.
Sifat sel myeloma :
- tidak mensintesis Ab.
- mutan (HGPRT-) — hypoxantin guanin
phosphoribosyl transferase.
- immortal.
4. Seleksi dengan HAT (hypoxantin Aminopterin
Thimidin). HAT mengandung aminopterin yang
menghambat sintesis nukleotida sel harus
mensintesis nukleotida melalui jalur alternatif
menggunakan enzim HGPRT.
* Sel myeloma mati dengan HAT karena HGPRT-.

* Sel B yang tidak berfusi mati secara alamiah.

* Sel B yang berfusi mempunyai sifat myeloma

(immortal) dan mempunyai sifat sel B (tidak
mati oleh HAT)  memproduksi mAb.
Manfaat mAb :

1. Identifikasi fenotip dengan melihat marker

spesifik pada permukaan sel.

mAb mengenal protein spesifik pada permukaan

sel yang diberi penanda fluorokhrom  dapat
mendeteksi protein/struktur spesifik pada
permukaan sel
- molekul CD pada permukaan limfosit.
- molekul TSA pada sel kanker.
2. Imunodiagnosis

Diagnosa penyakit infeksi dan sistemik ditegakkan

dgn menemukan/mendeteksi Ag atau Ab spesifik
dalam jaringan/sirkulasi menggunakan mAb dan
immunoassay.

3. Imunoterapi

Beberapa mAb telah dapat digunakan sebagai agen

untuk imunoterapi menetralkan toxin, virus, dan
tumor in vivo.
 GENETIC POLYMORPHISM

gy
 INTRODUCTION
• All population of plants, animals & Human
show normal variation
• Variation may be observed at :
- Phenotypic level (anatomical, Physiological,
biochemical, psychological)
- Genotypic level
. Variation may be :
- Continous
- Discontinous
 Introduction (cont.)

• Variation is normal natural phenomenon

- Many forms or variants in a given population
• Common form vs uncommon form
- Common  “normal” form/variant
- Uncommon  “abnormal” form/variant

• Advantage form vs disadvantage form

- Variant with advantage characters
- Variant with disadvantage character
- Variant with neutral character
 Introduction (cont.)
• Linked to natural selection  Darwin’s theory of
evolution :
- Principle of variation : morpology, physiology,
behavior
- Principle of heredity (offspring resemble their
parents more than those of unrelated individuals)
- Principle of selection (some forms are more
successful at surviving & reproducing than others
in a given environment)
 Definitions
• Polymorphism => derived form Greek means
“many forms”
• The simplest type called dimorphism
• A set of two or more common, alternative, normal
phenotypes
• Genetic polymorphism : the occurrence of two or
more alleles in which the frequency of the most
common allele is less than 99%
POLYMORPHISM GENETICS
DEFINITION
• Poly = > 1 ; Morphism = form

• Different Gene form = Allele

• >1 allelic gene = polymorphism
• mtDNA **, nDNA
• polymorphism detection by Restriction
enzymes
MITOCHONDRIAL DNA - mtDNA

• High DNA contain

• Replication autonomically
• Circle DNA : 15,7 – 19,5 kb
• High mutation rate
• 13 of protein genes
• Application : genetic population, ageing
process, deseases diagnostic etc.
 GENOME COMPLEXITY

NONCODING DNA :
• A large proportion of the DNA in eukaryotic
cells does not for protein
• Most genes have large intron seq. &
unknown function
•Much of this DNA consist of multiple
repeateds : short sequence element until
thousands copies
Why polymorphism ? (Source
of variation)
• Mutation

- a constant change in genetic material

- Occur at random from molecular to chromosomal
- Mutation rate varies among organism
• Recombination
 Genotype

Single locus Multi loci

Environmental factors 

PPhh en
e no
o t yy ppee

Protein, RNAs Biochemical/physiological/

(Biochemical genetic psychological/physical
Traits) characters
 MUTATION (in an individual)

Homozygote Heterozygote Hemizygote

Abnormal function/process Normal function/process

Genetic variant or
New allele in next generation
 MUTATION (in a population)

LETHAL NON-LETHAL

ACCEPTABLE PARTIALLY ACCEPTABLE

RARE ALLELE (s) POLYMORPHISM

 Types of variation

• Morphological variation

• Chromosomal polymorphism
• Immunological polymorphism
• Protein polymorphism
• DNA sequence polymorphism :
- Restriction site polymorphism
- Complete sequence polymorphism
 Use of Polymorphism

• Medical Forensic => Individual characteristic

• Disease/Environmental Related (predispo-
sition & tendency vs. resistance & advantage
vs disadvsantages)
• Genetic relationship (within & between
populations) => Molecular evolution
 ABO BLOOD GROUP & DISEASES

• Resistance of blood group O to epidemic

infectious diseases
• Susceptibility of blood group O to gastric &
peptic ulcer
• Lower risk of blood group O to rheumatic
fever
 HLA & DISEASES

• Strong association of HLA and

- IDDM
- Rheumatoid arthritis
- Pemphigus vulgaris
- Tuberculosis
- Leprosy etc
 GENETIC TRAITS &
CARDIOVASCULAR DISEASES

• HEART ATTCK * CANDIDATE GENES

- APOLIPOPROTEIN

• HYPERTENSION
- ENZYMES
- TRANSFER PROTEINS
- RECEPTOR
Marker DNA DNA Genom
Deteksi DNA dalam gel agarose pada UV
Transiluminator
 46

What’s So Great About

Molecular Diagnostics?
• As many as 5,000 diseases have direct genetic
causes
• High sensitivity and increased specificity for most
tests adds diagnostic utility
• Potential for simple standardized procedures an
automation
• rapid throughput
• Increased number of techniques for infectious
diseases
and tumor diagnostics
• A viable reflex for equivocal morphology
• Prices are falling