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MOLECULAR

DIAGNOSITICS

Prof . Fang Zheng,

Tianj in Medical Universi ty

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In t roduc t ion to

Molecu lar Diagnos t ics

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Outl ine

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 o f Molecu lar Diagnos t ics





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1. Molecular Diagnosis

Molecular diagnosis of human disorders

is referred to as the detection of the

various pathogenic mutations in DNAand /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 ofmolecular biology techniques

to expand scientific knowledge of the

natural history of diseases, identify

people who are at risk for acquiring

specific diseases, and diagnose humandiseases at the nucleic acid level.

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1. Molecular Diagnostics

Molecular diagnostics combines

laboratory medicine with the knowledge

and technology of molecular geneticsand has been enormously revolutionized

over the last decades, benefiting from the

discoveries in the field of molecular

biology.

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The information revolution in molecularbiology is permeating every aspect of

medical practice

The rate ofdisease gene discovery isincreasing exponentially, which facilitates

the understanding diseases at molecular

level

Molecular understanding of disease is

translated into diagnostic testing,

therapeutics, and eventually preventive

therapies

1. Molecular Diagnostics: Emergence

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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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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, determineappropriate treatment strategies, and

predict disease outcomes.

1. Molecular Diagnostics: Goal

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Histo ry o f Molecu lar Diagnos t ics




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2. 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 I n VitroAmplification of DNA (PCR)

2001 The Human Genome Project

Watson and Crick, Structure of DNA

The Molecular Biology Timeline

Sickle Cell Anemia Mutation1949

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Pauling introduced the term molecular

diseasein the medical vocabulary, based on

their discovery that a single amino acid changeleads to a sickle cell anem ia.

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.

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J.D. Watson and F.H.C. Crick(1953)A structure for deoxyribose nucleic acid.

Nature171:737

We wish to suggest a structure for thesalt 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

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


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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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1962 J. Watson & F. Crick: DNA structure

Max Perutz & John Kendrew: Protein sequence1958 Frederick Sanger: Insulin sequence

1980 Frederick Sanger: DNA sequencing

1984 Cesar Milstein & Georges Kohler: Monoclonal Ab


Laboratory of Molecular Biology(LMB) (Cavendish Laboratory )

1955- 12 scientistsreceived Noble Prize

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The first seeds of molecular diagnostics were provided

in the early days ofrecombinant DNA technology.

cDNA cloning and sequencing were invaluabletools for providing the basic knowledge on the primary

sequence of various genes.

DNA sequencingprovided a number of DNA probes,

allowing the analysis via southern blotting of genomic

regions, leading to the concept and application ofrestriction fragment length polymorphism (RELP) track amutant allele from heterozygous parents to a high-risk

pregnancy.

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The PCR Revolution

Kary Mull is

1985 41y

Invention o f PCR

1993 49yReceived 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 Revo lu t ion

PCR markedly decreased need for

radioactivity, allowed molecular diagnosticsto enter the clinical laboratory.

PCR either is used for the generation of DNA

fragments to be analyzed, or is part of thedetection methods

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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


Human Genome Project

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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


Human Genome Project

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Discovery of potential novel molecular

markers of human diseases

Identification of novel molecularmarkers of human diseases

Utility of molecular markers to develop

useful molecular assays for detection,diagnosis, and prediction of disease

outcomes

3. Impact on Human Diseases: Novelty

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3. Impact on Human Diseases: Advantage

Monitor diseases more accuratelyAllows for early treatment and betterpatient care

Determine most appropriate treatmentReduces or eliminates unnecessarytreatment

Reduces or eliminates inadequate

treatment

Yields greater cost effectiveness

Reduce patient morbidity and mortality

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Diagnostic-Identity of a disease

Prognostic-Outcome of a diseasePredictive-Possibility of a disease

Therapeutic-Response of adisease to treatment

3. Impact on Human Diseases: Practical

application

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HEMATOLOGY

INFECTIOUS

DISEASE

IDENTITYTESTING

GENETIC

DISEASE

SOLID

TUMORS

MolecularPathology

3. Impact on Human Diseases

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Molecular Genetics

Single gene disorders

Polygenic disorders

Chromosomal disorders


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Molecular Oncology

Diagnostic testing

Disease prognosis

Determination of predisposition


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Hematopathology

Diagnostic testing

Determination of clonality

Identity Testing

Parentage Clinical testing


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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 (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

Amplification Techniques

PCR polymerase chain reaction

LCR ligase chain reaction

NASBA nucleic-acid sequence-based amplification

Molecular Technologies in the Clinical Laboratory

DNA Sequencing

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Electrophoretic Methods

SSCP (single-strand conformation polymorphism)

DGGE (denaturing gradient gel electrophoresis)


Hybridization Techniques

Southern hybridization Blot

Northern hybridization Blot

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Biochip Technology

DNA micro-array

Protein micro-array


Recombinant DNA Technology

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4. Basis for Technology: Target specialty

Genetically-based diseases can bediagnosed

Specificity can be controlled

Single base changes can be detected

Expression of gene product is notrequired

Targets can be amplified >105

Nucleic acids are targeted by molecular assays

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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)

Diagnostic

Distinguishing variants of human disease based

on presence ofspecific molecular markers

(chromosome translocations in Burkitts

lymphoma: c-myc)

Understanding molecular pathogenesis of human

disease enables effective uti l ization of molecular assays

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Prognostic Prediction of likely patient outcomes based on

presence ofspecific molecular markers (gene

mutations predicting clinical course in cancer)



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Therapeutic Prediction of response to specific therapies

based on presence ofspecific molecular

markers (gene mutations predicting poordrug sensitivity inlung 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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Characterization of Gene Sequences

Facilitates characterization of disease-causingmutations

Molecular targets for assay development

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Completion of the sequence of the human

genome will enable identification of all

human genes and establishment ofdisease-gene relationships, facilitating

development of numerous new molecular

assays.

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Improvements in medicine

Microbial genome research

DNA forensics/identity

Improved agriculture and livestock

Better understanding of evolutionand human migration

More accurate risk assessment

Beneficial outcomes from human genome project

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Use of genetic information

Privacy/confidentiality

Psychological impact

Genetic testing

Reproductive options/issues

Education, standards, and quality control

Commercialization

Conceptual and philosophical implications

Human genome project: Ethical, Legal, and

Social Implications

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Whats So Great AboutMolecular Diagnostics?

As many as 5,000 diseases have direct genetic causes

High sensitivity and increased specificity for mosttests adds diagnostic utility

Potential for simple standardized procedures an

automation

rapid throughput

Increased number of techniques for infectious diseasesand tumor diagnostics

A viable reflex for equivocal morphology

Prices are falling

5. Conclusion

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The ultimate goal of the molecular diagnosticsis 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/orguidance toward appropriate and effective

treatment options.

5. Conclusion

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