MOLECULAR

DIAGNOSITICS

Prof. Fang Zheng,

Tianjin Medical University

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 Introduction to
Molecular Diagnostics

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Outline

Concept of Molecular Diagnostics

History of Molecular Diagnostics
Impact on Human Diseases
Basis for Molecular Assay
Management of the course

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Concept of Molecular Diagnostics
History of Molecular Diagnostics
Impact on Human Diseases
Basis for Molecular Assay
Management of the course

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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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1. Molecular Diagnostics

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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 1. Molecular Diagnostics

Molecular diagnostics combines

laboratory medicine with the knowledge
and technology of molecular genetics
and has been enormously revolutionized
over the last decades, benefiting from the
discoveries in the field of molecular
biology.
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1. Molecular Diagnostics: Emergence
The information revolution in molecular
biology is permeating every aspect of
medical practice
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
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1. Molecular Diagnostics: Significance

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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 1. Molecular Diagnostics: Goal

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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Concept of Molecular Diagnostics
History of Molecular Diagnostics
Impact on Human Diseases
Basis for Molecular Assay
Management of the course

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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
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2. History of Molecular Diagnostics
Sickle cell anemia
Sickle cell anemia is a genetic disease which
is caused by a single nucleotide change in the
6th aa of the -chain of hemoglobin.
 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
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Discovery of DNA Structure

J.D. Watson and F.H.C. Crick (1953) 16

 Discovery of DNA Structure

Rosalind E. Franklin
1920–1958
The structure of DNA was determined using X-ray
diffraction techniques. Much of the original X-ray
diffraction data was generated by Rosalind E. Franklin.
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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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2. History of Molecular Diagnostics
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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2. History of Molecular Diagnostics
The PCR Revolution

Kary Mullis
1985 41y
Invention of PCR
1993 49y
Received the Noble Prize

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2. History of Molecular Diagnostics
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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2. History of Molecular Diagnostics

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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2. History of Molecular Diagnostics
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; and
– To address the ethical, legal, and social issues
that arise from genome research
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2. History of Molecular Diagnostics
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; and
– To address the ethical, legal, and social issues
that arise from genome research
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Concept of Molecular Diagnostics
History of Molecular Diagnostics
Impact on Human Diseases
Basis for Molecular Assay
Management of the course

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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
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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
病种多，特定家系中发病率高，对群体影响小，
遗传性基因携带者的筛查

• Polygenic disorders
病种少，特定家系中发病率高，对群体影响大
遗传易感性的检测

• 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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Concept of Molecular Diagnostics
History of Molecular Diagnostics
Impact on Human Diseases
Basis for Molecular Assay
Management of the course

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4. Basis for Technology: Fundamental (1)

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多聚酶链反应
LCR ligase chain reaction 连接酶链反应
NASBA nucleic-acid sequence-based amplification
核酸序列依赖的扩增

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)
单链构象多态性
DGGE (denaturing gradient gel electrophoresis)
变性梯度凝胶电泳法

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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 (1)
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 (1)
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 (2)
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 (1)

Genetic Lesions in Human Disease

• Identification of genetic markers

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

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4. Basis for Molecular Assay: Molecular biology (1)

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 (2)

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 (4)
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 (5)
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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