MOLECULAR DIAGNOSITICS

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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
Sickle Cell Anemia Mutation
1949
Watson and Crick, Structure of DNA
1953
Recombinant DNA Technology
1970
DNA sequencing
1977
In Vitro Amplification of DNA (PCR)
1985
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 171737 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)
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Discovery of DNA Structure
Rosalind E. Franklin 19201958
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

• J. Watson F. Crick DNA structure
• Max Perutz John Kendrew Protein
  sequence
• Frederick Sanger Insulin sequence
• Frederick Sanger DNA sequencing
• 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
• (19902006)
• 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
• (19902006)
• 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
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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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• 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??????
• 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 gt105
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4. Basis for Molecular Assays Diseases
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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 Burkitts
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

• Whats 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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END