1 molecular diagnositics prof. fang zheng, tianjin medical university

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






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.

77

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.

88

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






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

1313


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

1414

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)


1717


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.

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……


Laboratory of Molecular Biology ，(LMB) (Cavendish Laboratory )

1955- 12 scientists received Noble Prize

2020


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.

2121


The PCR RevolutionThe PCR Revolution

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

2222



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.

2323



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


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


Human Genome ProjectHuman Genome Project

2626






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


3232

Molecular Oncology

• Diagnostic testing

• Disease prognosis

• Determination of predisposition


3333

Hematopathology• Diagnostic testing• Determination of clonality

Identity Testing• Parentage• Clinical testing


3434

Infectious Disease

• Qualitative and quantitative detection of infectious agents

• Microbial identity testing

• Genotyping/drug resistance testing


3535






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


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


Hybridization Techniques Southern hybridization Blot Northern hybridization Blot

3939


Biochip Technology DNA micro-array Protein micro-array


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


Prognostic• Prediction of likely patient outcomes based on

presence of specific molecular markers (gene

mutations predicting clinical course in cancer)


4444



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


Characterization of Gene Sequences

• Facilitates characterization of disease-causing mutations

• Molecular targets for assay development

4747


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


•• 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


•• 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

1 molecular diagnositics prof. fang zheng, tianjin medical university

Documents

molecular levelmolecular

molecular diagnositicsprof

term molecular disease

molecular assay management

field of molecular biology

useful molecular assays

human diseases basis

natural history of diseases