An introduction to

Molecular Diagnostics

Diploma

Dr. Basma Al- Sudani

Lecture 1

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1. Concept of Molecular Diagnostics
2. History of Molecular Diagnostics
3. Impact on Human Diseases
4. Basis for Molecular Assay

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 1. Molecular Diagnosis

• 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.
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• The use of molecular biology techniques to
expand scientific knowledge of the natural
history of diseases, identify people who are
at risk for acquiring specific diseases, and
diagnose human diseases at the nucleic acid
level.

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• Molecular diagnostics combines
laboratory medicine with the
knowledge and technology of
molecular genetics.

• It has been revolutionized over the

last decades, benefiting from the
discoveries in the field of molecular
biology.
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a. Molecular Diagnostics: Emerging trends

• The rate of disease gene discovery is

increasing exponentially, which facilitates
the understanding diseases at molecular
level

• Molecular understanding of disease is

translated into diagnostic testing,
therapeutics, and eventually preventive
therapies 6
b. Molecular Diagnostics: Significance in
Human medicine
• To face the new century, the medical
practitioner not only understand
molecular biology, but must also embrace
the use of this rapidly expanding body of
information in his medical practice,
whether practicing family medicine,
oncology, obstetrics and gynecology,
pathology, or any other medical specialty.
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 c. Molecular Diagnostics: Goal

Based on
•To introduce essential concepts in molecular
diagnostics that impact on the identification
of novel markers of human diseases
•To develop and apply useful molecular
assays to monitor disease, determine
appropriate treatment strategies, and predict
disease outcomes.
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2. History of Molecular Diagnostics
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
2005-11 Sequencing technologies and Genome sequencing
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 Sickle cell anemia

Sickle cell anemia is a genetic disease which is

caused by a single nucleotide change in the 6th of
the -chain of hemoglobin.
 Pauling introduced the term molecular disease in
the medical vocabulary, based on their discovery
that a single amino acid change leads to a sickle cell
anemia.

 In principle, their findings have set the foundations

of molecular diagnostics.
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Sickle Cell Anemia

Figure A. Normal red blood cells

flowing freely in a blood vessel. The
inset image shows a cross-section of
a normal red blood cell with normal
hemoglobin.

Figure B. Abnormal, sickled red

blood cells clumping and blocking
blood flow in a blood vessel. The
inset image shows a cross-section of
a sickle cell with abnormal
hemoglobin.

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 Discovery of DNA Structure

J.D. Watson and F.H.C. Crick (1953)

A structure for deoxyribose nucleic acid.
Nature 171:737

“We wish to suggest a structure for the

salt of deoxyribose nucleic acid (D.N.A.).
This structure has novel features which
are of considerable biological interest.”

One of the most important biological discovery

in the 20th century 12
 Discovery of DNA Structure

J.D. Watson and F.H.C. Crick (1953)

Rosalind E. Franklin
1920–1958
The structure of DNA was determined using X-ray diffraction techniques. Much of13
the
original X-ray diffraction data was generated by Rosalind E. Franklin.
 Discovery of DNA Structure

Laboratory of Molecular Biology ，

(LMB) (Cavendish Laboratory )
1955- 12 scientists
received Noble Prize
1962 J. Watson & F. Crick: DNA structure
Max Perutz & John Kendrew: Protein sequence
1958 Frederick Sanger: Insulin sequence
1980 Frederick Sanger: DNA sequencing
1984 Cesar Milstein & Georges Kohler: Monoclonal Ab

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The first seeds of molecular diagnostics were provided in the
early days of recombinant DNA technology.
cDNA cloning and sequencing were invaluable tools for
providing the basic knowledge on the primary sequence of
various genes.
DNA sequencing provided a number of DNA probes,
allowing the analysis via southern blotting of genomic regions,
leading to the concept and application of restriction fragment
length polymorphism (RELP) track a mutant allele from
heterozygous parents to a high-risk pregnancy.

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 The PCR Revolution

Kary Mullis
1985
Invention of PCR
1993
Received the Noble Prize

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 The PCR Revolution

• PCR has greatly facilitated and revolutionized

molecular diagnostics.

• Its most powerful feature - large amount of copies of

the target sequence generated by its exponential
amplification, which allows the identification of a
known mutation within a single day.
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 The PCR Revolution
• PCR markedly decreased need for radioactivity,
allowed molecular diagnostics to enter the clinical
laboratory.

• PCR either is used for the generation of DNA

fragments to be analyzed, or is part of the
detection methods

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 Human Genome Project

• U.S. Government project coordinated by the Dept. of

Energy and NIH
• Goals of the Human Genome Project
(1990–2006)
– To identify all of the genes in human DNA
– To determine the sequences of the 3 billion bases that
make up human DNA
– To create databases
– To develop tools for data analysis
– To address the ethical, legal, and social issues that arise
from genome research 19
 3. Impact on Human Diseases: Novelty

• Discovery of potential novel molecular

markers of human diseases
• Identification of novel molecular markers of
human diseases
• Utility of molecular markers to develop
useful molecular assays for detection,
diagnosis, and prediction of disease
outcomes
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 3. Impact on Human Diseases: Advantage

• Monitor diseases more accurately

Allows for early treatment and better patient
care

• Determine most appropriate treatment

Reduces or eliminates unnecessary treatment
Reduces or eliminates inadequate treatment
Yields greater cost effectiveness

• Reduce patient morbidity and mortality 21

 3. 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

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 3. Impact on Human Diseases

INFECTIOUS
DISEASE
HEMATOLOGY

Molecular
Pathology

SOLID
IDENTITY TUMORS
TESTING

GENETIC
DISEASE

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 3. Impact on Human Diseases

Molecular Genetics
• Single gene disorders

• Polygenic disorders

• Chromosomal disorders

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 3. Impact on Human Diseases

Molecular Oncology

• Diagnostic testing
• Disease prognosis
• Determination of predisposition

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3. Impact on Human Diseases

Hematopathology
• Diagnostic testing
• Determination of clonality

Identity Testing
• Parentage
• Clinical testing

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3. Impact on Human Diseases

 Infectious Disease
• Qualitative and quantitative
detection of infectious agents
• Microbial identity testing
• Genotyping/drug resistance
testing

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 4. Basis for Technology: Fundamental

Advance in the understanding of the

structure and chemistry of nucleic acids
have facilitated the development of
technologies that can be employed
effectively in molecular diagnostics.

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 4. Basis for Technology: Platform
Molecular Technologies in the Clinical Laboratory

Amplification Techniques
PCR polymerase chain reaction
LCR ligase chain reaction
NASBA nucleic-acid sequence-based amplification

DNA Sequencing

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 4. Basis for Technology: Platform
Molecular Technologies in the Clinical Laboratory

Hybridization Techniques
Southern hybridization Blot
Northern hybridization Blot

Electrophoretic Methods
SSCP (single-strand conformation polymorphism)
DGGE (denaturing gradient gel electrophoresis)
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 4. Basis for Technology: Platform
Molecular Technologies in the Clinical Laboratory

Recombinant DNA Technology

Biochip Technology
DNA micro-array
Protein micro-array

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4. Basis for Technology: Target specialty
Nucleic acids are targeted by molecular assays
• Genetically-based diseases can be
diagnosed
• Specificity can be controlled
• Single base changes can be detected
• Expression of gene product is not
required
• Targets can be amplified >105
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4. Basis for Molecular Assays: Diseases
Cause (etiology)

Mechanism (pathogenesis)

Structural alterations (morphologic/molecular)

Functional consequences (clinical significance)

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 4. Basis for Molecular Assay: Pathogenesis
Understanding molecular pathogenesis of human
disease enables effective utilization of molecular assays

Diagnostic
• Distinguishing variants of human disease based
on presence of specific molecular markers
(chromosome translocations in Burkitt’s
lymphoma: c-myc)

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 4. Basis for Molecular Assay: Pathogenesis

Understanding molecular pathogenesis of human

disease enables effective utilization of molecular assays

Prognostic
• Prediction of likely patient outcomes based on
presence of specific molecular markers (gene
mutations predicting clinical course in cancer)

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 4. Basis for Molecular Assay: Pathogenesis
Understanding molecular pathogenesis of human
disease enables effective utilization of molecular assays

Therapeutic
• Prediction of response to specific therapies
based on presence of specific molecular
markers (gene mutations predicting poor
drug sensitivity in lung cancer: p53, k-ras)

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 4. Basis for Molecular Assay: Molecular biology

 Genetic Lesions in Human Disease

• Identification of genetic markers

• Identification of disease-related genes
• Molecular targets for assay development

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 Characterization of Gene Sequences

• Facilitates characterization of disease-causing

mutations
• Molecular targets for assay development

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 4. Basis for Molecular Assay: Molecular biology

Completion of the sequence of the human

genome will enable identification of all
human genes and establishment of
disease-gene relationships, facilitating
development of numerous new molecular
assays.

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 4. Basis for Molecular Assay: Molecular biology
Beneficial outcomes from human genome project
• Improvements in medicine
• Microbial genome research
• DNA forensics/identity
• Improved agriculture and livestock
• Better understanding of evolution and
human migration
• More accurate risk assessment
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4. Basis for Molecular Assay: Molecular biology
Human genome project: Ethical, Legal, and
Social Implications

• Use of genetic information

• Privacy/confidentiality
• Psychological impact
• Genetic testing
• Reproductive options/issues
• Education, standards, and quality control
• Commercialization
• Conceptual and philosophical implications
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5. Conclusion
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
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5. Conclusion

The ultimate goal of the molecular diagnostics

is to provide molecular information that will
combine with and complement information
related to patient history and symptomology,
clinical laboratory results, histopathological
findings, and other diagnostic information to
provide a more sensitive, precise, and accurate
determination of disease diagnosis and/or
guidance toward appropriate and effective
treatment options.
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