Molecular techniques.ppt
- 1. 1 An introduction to Molecular Diagnostics Lecture 1
- 2. 2 1. Concept of Molecular Diagnostics 2. History of Molecular Diagnostics 3. Impact on Human Diseases 4. Basis for Molecular Assay
- 3. 3 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.
- 4. 4 • 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.
- 5. 5 • 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.
- 6. 6 • 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 a. Molecular Diagnostics: Emerging trends
- 7. 7 b. 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.
- 8. 8 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. c. Molecular Diagnostics: Goal
- 9. 9 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 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 Sequencing technologies and Genome sequencing 2005-11
- 10. 10 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 of the -chain of hemoglobin. Sickle cell anemia
- 11. 11 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.
- 12. 12 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
- 13. 13 J.D. Watson and F.H.C. Crick (1953) Discovery of DNA Structure 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.
- 14. 14 1962 J. Watson & F. Crick: DNA structure Max Perutz & John Kendrew: Protein sequence 1958 Frederick Sanger: Insulin sequence 1980 Frederick Sanger: DNA sequencing 1984 Cesar Milstein & Georges Kohler: Monoclonal Ab Discovery of DNA Structure Laboratory of Molecular Biology， (LMB) (Cavendish Laboratory ) 1955- 12 scientists received Noble Prize
- 15. 15 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.
- 16. 16 The PCR Revolution Kary Mullis 1985 Invention of PCR 1993 Received the Noble Prize
- 17. 17 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.
- 18. 18 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
- 19. 19 • 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 – To address the ethical, legal, and social issues that arise from genome research Human Genome Project
- 20. 20 • 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 3. Impact on Human Diseases: Novelty
- 21. 21 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
- 22. 22 • Diagnostic-Identity of a disease • Prognostic-Outcome of a disease • Predictive-Possibility of a disease • Therapeutic-Response of a disease to treatment 3. Impact on Human Diseases: Practical application
- 23. 23 HEMATOLOGY INFECTIOUS DISEASE IDENTITY TESTING GENETIC DISEASE SOLID TUMORS Molecular Pathology 3. Impact on Human Diseases
- 24. 24 Molecular Genetics • Single gene disorders • Polygenic disorders • Chromosomal disorders 3. Impact on Human Diseases
- 25. 25 Molecular Oncology • Diagnostic testing • Disease prognosis • Determination of predisposition 3. Impact on Human Diseases
- 26. 26 Hematopathology • Diagnostic testing • Determination of clonality Identity Testing • Parentage • Clinical testing 3. Impact on Human Diseases
- 27. 27 Infectious Disease • Qualitative and quantitative detection of infectious agents • Microbial identity testing • Genotyping/drug resistance testing 3. Impact on Human Diseases
- 28. 28 4. Basis for Technology: Fundamental 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.
- 29. 29 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
- 30. 30 4. Basis for Technology: Platform Electrophoretic Methods SSCP (single-strand conformation polymorphism) DGGE (denaturing gradient gel electrophoresis) Molecular Technologies in the Clinical Laboratory Hybridization Techniques Southern hybridization Blot Northern hybridization Blot
- 31. 31 4. Basis for Technology: Platform Biochip Technology DNA micro-array Protein micro-array Molecular Technologies in the Clinical Laboratory Recombinant DNA Technology
- 32. 32 4. Basis for Technology: Target 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
- 33. 33 4. Basis for Molecular Assays: Diseases Cause (etiology) Mechanism (pathogenesis) Structural alterations (morphologic/molecular) Functional consequences (clinical significance)
- 34. 34 4. Basis for Molecular Assay: Pathogenesis 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
- 35. 35 4. Basis for Molecular Assay: Pathogenesis 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
- 36. 36 4. Basis for Molecular Assay: Pathogenesis 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)
- 37. 37 4. Basis for Molecular Assay: Molecular biology Genetic Lesions in Human Disease • Identification of genetic markers • Identification of disease-related genes • Molecular targets for assay development
- 38. 38 Characterization of Gene Sequences • Facilitates characterization of disease-causing mutations • Molecular targets for assay development
- 39. 39 4. Basis for Molecular Assay: Molecular biology 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.
- 40. 40 4. Basis for Molecular Assay: Molecular biology • Improvements in medicine • Microbial genome research • DNA forensics/identity • Improved agriculture and livestock • Better understanding of evolution and human migration • More accurate risk assessment Beneficial outcomes from human genome project
- 41. 41 4. Basis for Molecular Assay: Molecular biology • 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
- 42. 42 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
- 43. 43 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