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11
MOLECULAR MOLECULAR DIAGNOSITICSDIAGNOSITICS
Prof. Prof. Fang Zheng, Fang Zheng,
Tianjin Medical UniversityTianjin Medical University
22
Introduction toIntroduction to
Molecular DiagnosticsMolecular Diagnostics
33
Outline
Concept of Molecular Diagnostics
History of Molecular Diagnostics
Impact on Human Diseases
Basis for Molecular Assay
Management of the course
44
Concept of Molecular Diagnostics
History of Molecular Diagnostics
Impact on Human Diseases
Basis for Molecular Assay
Management of the course
55
1. Molecular Diagnosis1. 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.
66
1. Molecular Diagnost1. Molecular Diagnosticsics
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 Diagnostics1. 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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The The information revolutioninformation revolution in molecular in molecular biology is permeating every aspect of biology is permeating every aspect of medical practicemedical practice
The rate of The rate of disease gene discoverydisease gene discovery is is increasing exponentially, which facilitates increasing exponentially, which facilitates the understanding diseases at molecular the understanding diseases at molecular levellevel
Molecular understandingMolecular understanding of disease is of disease is translated into diagnostic testing, translated into diagnostic testing, therapeutics, and eventually preventive therapeutics, and eventually preventive therapiestherapies
1. Molecular Diagnostics: 1. Molecular Diagnostics: EmergenceEmergence
99
1. Molecular Diagnostics: 1. Molecular Diagnostics: SignificanceSignificance
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.
1010
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.
1. Molecular Diagnostics: Goal1. Molecular Diagnostics: Goal
1111
Concept of Molecular Diagnostics
History of Molecular Diagnostics
Impact on Human Diseases
Basis for Molecular Assay
Management of the course
1212
2. History of Molecular Diagnostics2. History of Molecular Diagnostics
1865 Gregor Mendel, Law of Heredity
1866 Johann Miescher, Purification of DNA
1953
1970 Recombinant DNA Technology
1977 DNA sequencing
1985 In Vitro Amplification of DNA (PCR)
2001 The Human Genome Project
Watson and Crick, Structure of DNA
The Molecular Biology Timeline
Sickle Cell Anemia Mutation 1949
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2. History of Molecular Diagnostics2. History of Molecular Diagnostics
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.
Sickle cell anemia is a genetic disease which is caused by a single nucleotide change in the 6th aa of the -chain of hemoglobin.
Sickle cell anemia
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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.
1515
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.”
Discovery of DNA Structure
One of the most important biological discovery in the 20th century
1616J.D. Watson and F.H.C. Crick (1953)
Discovery of DNA Structure
1717
Discovery of DNA Structure
Rosalind E. Franklin1920–1958
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.
1818
1919
1962 J. Watson & F. Crick: DNA structure Max Perutz & John Kendrew: Protein sequence1958 Frederick Sanger: Insulin sequence 1980 Frederick Sanger: DNA sequencing1984 Cesar Milstein & Georges Kohler: Monoclonal Ab……
Discovery of DNA Structure
Laboratory of Molecular Biology ,(LMB) (Cavendish Laboratory )
1955- 12 scientists received Noble Prize
2020
2. History of Molecular Diagnostics2. 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 Diagnostics2. History of Molecular Diagnostics
The PCR RevolutionThe PCR Revolution
Kary Mullis 1985 41y Invention of PCR 1993 49y Received the Noble Prize
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2. History of Molecular Diagnostics2. History of Molecular Diagnostics
The PCR RevolutionThe 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 Diagnostics2. History of Molecular Diagnostics
The PCR RevolutionThe 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
2424
•• U.S. Government project coordinated by the Dept. of U.S. Government project coordinated by the Dept. of Energy and NIH Energy and NIH
•• Goals of the Human Genome Project Goals of the Human Genome Project
(1990–2006)
– – To identify all of the genes in human DNA;To identify all of the genes in human DNA;
– – To determine the sequences of the 3 billion bases To determine the sequences of the 3 billion bases that make up human DNA;that make up human DNA;
– – To create databases;To create databases;
– – To develop tools for data analysis; andTo develop tools for data analysis; and
– – To address the ethical, legal, and social issues To address the ethical, legal, and social issues that arise from genome researchthat arise from genome research
2. History of Molecular Diagnostics2. History of Molecular Diagnostics
Human Genome ProjectHuman Genome Project
2525
•• U.S. Government project coordinated by the Dept. of U.S. Government project coordinated by the Dept. of Energy and NIH Energy and NIH
•• Goals of the Human Genome Project Goals of the Human Genome Project
(1990–2006)
– – To identify all of the genes in human DNA;To identify all of the genes in human DNA;
– – To determine the sequences of the 3 billion bases To determine the sequences of the 3 billion bases that make up human DNA;that make up human DNA;
– – To create databases;To create databases;
– – To develop tools for data analysis; andTo develop tools for data analysis; and
– – To address the ethical, legal, and social issues To address the ethical, legal, and social issues that arise from genome researchthat arise from genome research
2. History of Molecular Diagnostics2. History of Molecular Diagnostics
Human Genome ProjectHuman Genome Project
2626
Concept of Molecular Diagnostics
History of Molecular Diagnostics
Impact on Human Diseases
Basis for Molecular Assay
Management of the course
2727
DiscoveryDiscovery of potential novel molecular of potential novel molecular markers of human diseases markers of human diseases
IdentificationIdentification of novel molecular of novel molecular markers of human diseases markers of human diseases
Utility Utility of molecular markers to develop of molecular markers to develop useful molecular assays for detection, useful molecular assays for detection, diagnosis, and prediction of disease diagnosis, and prediction of disease outcomesoutcomes
3. Impact on Human Diseases: Novelty 3. Impact on Human Diseases: Novelty
2828
3. Impact on Human Diseases: 3. Impact on Human Diseases: AdvantageAdvantage
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
2929
Diagnostic-Identity of a diseaseDiagnostic-Identity of a disease
Prognostic-Outcome of a diseasePrognostic-Outcome of a disease
Predictive-Possibility of a diseasePredictive-Possibility of a disease
Therapeutic-Response of a Therapeutic-Response of a disease to treatmentdisease to treatment
3. Impact on Human Diseases: Practical 3. Impact on Human Diseases: Practical applicationapplication
3030
HEMATOLOGY
INFECTIOUS DISEASE
IDENTITYTESTING
GENETICDISEASE
SOLIDTUMORS
MolecularPathology
3. Impact on Human Diseases3. Impact on Human Diseases
3131
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
3. Impact on Human Diseases3. Impact on Human Diseases
3232
Molecular Oncology
• Diagnostic testing
• Disease prognosis
• Determination of predisposition
3. Impact on Human Diseases3. Impact on Human Diseases
3333
Hematopathology• Diagnostic testing• Determination of clonality
Identity Testing• Parentage• Clinical testing
3. Impact on Human Diseases3. Impact on Human Diseases
3434
Infectious Disease
• Qualitative and quantitative detection of infectious agents
• Microbial identity testing
• Genotyping/drug resistance testing
3. Impact on Human Diseases3. Impact on Human Diseases
3535
Concept of Molecular Diagnostics
History of Molecular Diagnostics
Impact on Human Diseases
Basis for Molecular Assay
Management of the course
3636
4. Basis for Technology: 4. Basis for Technology: Fundamental (1)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.
3737
4. Basis for Technology: 4. Basis for Technology: PlatformPlatform
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
Molecular Technologies in the Clinical Laboratory
DNA Sequencing
3838
4. Basis for Technology: 4. Basis for Technology: PlatformPlatform
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
Molecular Technologies in the Clinical Laboratory
Hybridization Techniques Southern hybridization Blot Northern hybridization Blot
3939
4. Basis for Technology: 4. Basis for Technology: PlatformPlatform
Biochip Technology DNA micro-array Protein micro-array
Molecular Technologies in the Clinical Laboratory
Recombinant DNA Technology
4040
4. Basis for Technology: 4. Basis for Technology: Target specialtyTarget specialty
• 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
Nucleic acids are targeted by molecular assays
4141
4. Basis for Molecular Assays: 4. Basis for Molecular Assays: DiseasesDiseases
Cause (etiology)
Mechanism (pathogenesis)
Structural alterations (morphologic/molecular)
Functional consequences (clinical significance)
4242
4. Basis for Molecular Assay: 4. Basis for Molecular Assay: Pathogenesis (1)Pathogenesis (1)
Diagnostic• Distinguishing variants of human disease based
on presence of specific molecular markers
(chromosome translocations in Burkitt’s
lymphoma: c-myc)
Understanding molecular pathogenesis of human disease enables effective utilization of molecular assays
4343
4. Basis for Molecular Assay: 4. Basis for Molecular Assay: Pathogenesis (1)Pathogenesis (1)
Prognostic• Prediction of likely patient outcomes based on
presence of specific molecular markers (gene
mutations predicting clinical course in cancer)
Understanding molecular pathogenesis of human disease enables effective utilization of molecular assays
4444
4. Basis for Molecular Assay: 4. Basis for Molecular Assay: Pathogenesis (2)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)
4545
4. Basis for Molecular Assay:4. Basis for Molecular Assay: Molecular biology Molecular biology (1)(1)
Genetic Lesions in Human Disease
• Identification of genetic markers
• Identification of disease-related genes
• Molecular targets for assay development
4646
4. Basis for Molecular Assay:4. Basis for Molecular Assay: Molecular biology Molecular biology (1)(1)
Characterization of Gene Sequences
• Facilitates characterization of disease-causing mutations
• Molecular targets for assay development
4747
4. Basis for Molecular Assay:4. Basis for Molecular Assay: Molecular biology Molecular biology (2)(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.
4848
4. Basis for Molecular Assay:4. Basis for Molecular Assay: Molecular biology Molecular biology (4)(4)
•• Improvements in medicineImprovements in medicine
•• Microbial genome research Microbial genome research
•• DNA forensics/identityDNA forensics/identity
•• Improved agriculture and livestockImproved agriculture and livestock
•• Better understanding of evolution Better understanding of evolution and human migrationand human migration
•• More accurate risk assessmentMore accurate risk assessment
Beneficial outcomes from human genome project
4949
4. Basis for Molecular Assay:4. Basis for Molecular Assay: Molecular biology Molecular biology (5)(5)
•• Use of genetic informationUse of genetic information
•• Privacy/confidentialityPrivacy/confidentiality
•• Psychological impactPsychological impact
•• Genetic testingGenetic testing
•• Reproductive options/issuesReproductive options/issues
•• Education, standards, and quality controlEducation, standards, and quality control
•• CommercializationCommercialization
•• Conceptual and philosophical implicationsConceptual and philosophical implications
Human genome project: Ethical, Legal, and Social Implications
5050
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
5. Conclusion
5151
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.
5. Conclusion
5252
ENDEND