1、1,MOLECULAR DIAGNOSITICS,Prof. Fang Zheng, Tianjin Medical University,2,Introduction to Molecular Diagnostics,3,Outline,Concept of Molecular Diagnostics History of Molecular Diagnostics Impact on Human Diseases Basis for Molecular Assay Management of the course,4,Concept of Molecular Diagnostics His
2、tory of Molecular Diagnostics Impact on Human Diseases Basis for Molecular Assay Management of the course,5,1. 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, d
3、iagnosis, sub-classification, prognosis, and monitoring response to therapy.,6,1. 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 disea
4、ses at the nucleic acid level.,7,1. 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.,8,The
5、information revolution in molecular biology is permeating every aspect of medical practiceThe rate of disease gene discovery is increasing exponentially, which facilitates the understanding diseases at molecular levelMolecular understanding of disease is translated into diagnostic testing, therapeut
6、ics, and eventually preventive therapies,1. Molecular Diagnostics: Emergence,9,1. 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 pr
7、actice, whether practicing family medicine, oncology, obstetrics and gynecology, pathology, or any other medical specialty.,10,To introduce essential concepts in molecular diagnostics that impact on the identification of novel markers of human diseasesTo develop and apply useful molecular assays to
8、monitor disease, determine appropriate treatment strategies, and predict disease outcomes.,1. Molecular Diagnostics: Goal,11,Concept of Molecular Diagnostics History of Molecular Diagnostics Impact on Human Diseases Basis for Molecular Assay Management of the course,12,2. History of Molecular Diagno
9、stics,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 Mut
10、ation,1949,13,2. 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 ce
11、ll 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,14,Sickle Cell AnemiaFigure 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 nor
12、mal 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.,15,J.D. Watson and F.H.C. Crick (1953)A structure for deoxyribose nucleic acid.Nature 171:737“We wish to su
13、ggest 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,16,J.D. Watson and F.H.C. Crick (1953),Discovery of DNA
14、 Structure,17,Discovery of DNA Structure,Rosalind E. Franklin19201958,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.,18,19,J. Watson & F. Crick: DNA structure Max Perutz & John Kendrew: Protei
15、n sequence FrederickSanger: Insulin sequence FrederickSanger: DNA sequencing Cesar Milstein & Georges Kohler: Monoclonal Ab,Discovery of DNA Structure,Laboratory of MolecularBiology，(LMB) (Cavendish Laboratory ),1955- 12 scientists received Noble Prize,20,2. History of Molecular Diagnostics,The firs
16、t 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 sout
17、hern 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.,21,2. History of Molecular Diagnostics,The PCR Revolution,Kary Mullis 1985 41y Invention of PCR 1993 4
18、9y Received the Noble Prize,22,2. History of Molecular Diagnostics,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 identific
19、ation of a known mutation within a single day.,23,2. History of Molecular Diagnostics,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
20、 the detection methods,24,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 dat
21、a analysis; and To address the ethical, legal, and social issues that arise from genome research,2. History of Molecular Diagnostics,Human Genome Project,25,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
22、 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,2. History of Molecular Diagnostics,Human Genome Project,26,Concept of M
23、olecular Diagnostics History of Molecular Diagnostics Impact on Human Diseases Basis for Molecular Assay Management of the course,27,Discovery of potential novel molecular markers of human diseases Identification of novel molecular markers of human diseases Utility of molecular markers to develop us
24、eful molecular assays for detection, diagnosis, and prediction of disease outcomes,3. Impact on Human Diseases: Novelty,28,3. Impact on Human Diseases: Advantage,Monitor diseases more accurately Allows for early treatment and better patient careDetermine most appropriate treatment Reduces or elimina
25、tes unnecessary treatment Reduces or eliminates inadequate treatment Yields greater cost effectivenessReduce patient morbidity and mortality,29,Diagnostic-Identity of a disease Prognostic-Outcome of a disease Predictive-Possibility of a disease Therapeutic-Response of a disease to treatment,3. Impac
26、t on Human Diseases: Practical application,30,3. Impact on Human Diseases,31,Molecular GeneticsSingle 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 Diseases,32,Molecular OncologyDiagnostic testingDisease prog
27、nosisDetermination of predisposition,3. Impact on Human Diseases,33,HematopathologyDiagnostic testingDetermination of clonalityIdentity Testing Parentage Clinical testing,3. Impact on Human Diseases,34,Infectious DiseaseQualitative and quantitative detection of infectious agentsMicrobial identity te
28、stingGenotyping/drug resistance testing,3. Impact on Human Diseases,35,Concept of Molecular Diagnostics History of Molecular Diagnostics Impact on Human Diseases Basis for Molecular Assay Management of the course,36,4. Basis for Technology: Fundamental (1),Advance in the understanding of the structu
29、re and chemistry of nucleic acids have facilitated the development of technologies that can be employed effectively in molecular diagnostics.,37,4. Basis for Technology: Platform,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
30、-based amplification 核酸序列依赖的扩增,Molecular Technologies in the Clinical Laboratory,DNA Sequencing,38,4. Basis for Technology: Platform,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 C
31、linical Laboratory,Hybridization Techniques Southern hybridization Blot Northern hybridization Blot,39,4. Basis for Technology: Platform,Biochip Technology DNA micro-array Protein micro-array,Molecular Technologies in the Clinical Laboratory,Recombinant DNA Technology,40,4. Basis for Technology: Tar
32、get 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,41,4. Basis for Molecular Assays: Diseases,42,4. Basis fo
33、r Molecular Assay: Pathogenesis (1),DiagnosticDistinguishing variants of human disease based on presence of specific molecular markers (chromosome translocations in Burkitts lymphoma: c-myc),Understanding molecular pathogenesis of human disease enables effective utilization of molecular assays,43,4.
34、 Basis for Molecular Assay: Pathogenesis (1),PrognosticPrediction 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,4
35、4,4. Basis for Molecular Assay: Pathogenesis (2),Understanding molecular pathogenesis of human disease enables effective utilization of molecular assays,TherapeuticPrediction of response to specific therapies based on presence of specific molecular markers (gene mutations predicting poor drug sensit
36、ivity in lung cancer: p53, k-ras),45,4. Basis for Molecular Assay: Molecular biology (1),Genetic Lesions in Human DiseaseIdentification of genetic markersIdentification of disease-related genesMolecular targets for assay development,46,4. Basis for Molecular Assay: Molecular biology (1),Characteriza
37、tion of Gene SequencesFacilitates characterization of disease-causing mutationsMolecular targets for assay development,47,4. 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
38、 relationships, facilitating development of numerous new molecular assays.,48,4. Basis for Molecular Assay: Molecular biology (4),Improvements in medicineMicrobial genome research DNA forensics/identityImproved agriculture and livestockBetter understanding of evolution and human migrationMore accura
39、te risk assessment,Beneficial outcomes from human genome project,49,4. Basis for Molecular Assay: Molecular biology (5),Use of genetic informationPrivacy/confidentialityPsychological impactGenetic testingReproductive options/issuesEducation, standards, and quality controlCommercializationConceptual
40、and philosophical implications,Human genome project: Ethical, Legal, and Social Implications,50,Whats 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 stan
41、dardized 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,51,The ultimate goal of the molecular diagnostics is to provide molecular information that will co
42、mbine 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,52,END,
