1. Molecular Diagnostics Techniques
Lecture 1:
An introduction to
Molecular Diagnostics Techniques
I. Basics of Molecular Biology:
DNA, RNA, Protein, Transcription, translation, Genome
II. Basic Tools used in Molecular Biology:
PCR, Electrophoresis
III. Molecular markers
Type I and type II genetic markers
IV. PRINCIPLES OF DNA ISOLATION & PURIFICATION
protocols

2. Molecular Diagnostics Techniques
Lecture 2:
An introduction to
Molecular Diagnostics Techniques
1. Concept of Molecular Diagnostics
2. History of Molecular Diagnostics
3. Impact on Human Diseases
4. Basis for Molecular Assay

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

4. 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.

5. 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.

6. 1. Molecular Diagnostics
Molecular diagnostics combines laboratory medicine with the knowledge and technology of molecular genetics
It has been revolutionized over the last decades, benefiting from the discoveries in the field of molecular biology.

7. 1. Molecular Diagnostics: Emerging trends
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

8. 1. Molecular Diagnostics: Significance in Human medicine
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.

9. 1. Molecular Diagnostics: Goal
Based on
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.

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

11. 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
Sequencing technologies and Genome sequencing

12. Sickle cell anemia 2. History 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.
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.

13. 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.

14. One of the most important biological discovery in the 20th century
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

15. J.D. Watson and F.H.C. Crick (1953)
Discovery of DNA Structure
J.D. Watson and F.H.C. Crick (1953)
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.

16. Discovery of DNA Structure
Laboratory of Molecular Biology，(LMB) (Cavendish  Laboratory )
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 ……

17. 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.

18. The PCR Revolution 2. History of Molecular Diagnostics Kary Mullis
1985
Invention of PCR
1993
Received the Noble Prize

19. The PCR Revolution 2. History of Molecular Diagnostics
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.

20. The PCR Revolution 2. History of Molecular Diagnostics
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

21. 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

22. 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

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

24. 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

25. 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

26. 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

27. 3. Impact on Human Diseases
HEMATOLOGY
INFECTIOUS
DISEASE
IDENTITY
TESTING
GENETIC
SOLID
TUMORS
Molecular
Pathology

28. 3. Impact on Human Diseases
Molecular Genetics
• Single gene disorders
• Polygenic disorders
• Chromosomal disorders

29. 3. Impact on Human Diseases
Molecular Oncology
• Diagnostic testing
• Disease prognosis
• Determination of predisposition

30. 3. Impact on Human Diseases
Hematopathology
• Diagnostic testing
• Determination of clonality
Identity Testing
• Parentage
• Clinical testing

31. 3. Impact on Human Diseases
Infectious Disease
• Qualitative and quantitative detection of infectious agents
• Microbial identity testing
• Genotyping/drug resistance testing

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

33. 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.

34. 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

35. 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)

36. 4. Basis for Technology: Platform
Molecular Technologies in the Clinical Laboratory
Recombinant DNA Technology
Biochip Technology
DNA micro-array
Protein micro-array

37. 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

38. 4. Basis for Molecular Assays: Diseases
Cause (etiology)
Mechanism (pathogenesis)
Structural alterations (morphologic/molecular)
Functional consequences (clinical significance)

39. 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)

40. 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)

41. 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)

42. 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

43. 4. Basis for Molecular Assay: Molecular biology (1)
Characterization of Gene Sequences
• Facilitates characterization of disease-causing mutations
• Molecular targets for assay development

44. 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.

45. 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

46. 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

47. Molecular Diagnostics?
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

48. 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.