MET Reference Book : Molecular Diagnostics: Fundamentals, Methods and Clinical Applications (Lela Buckingham, Maribeth L.
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1 MET 4217 Reference Book : Molecular Diagnostics: Fundamentals, Methods and Clinical Applications (Lela Buckingham, Maribeth L. Flaws, ) ISBN-13: ISBN-10: / Applied Medical Sciences ( 1
2 A. Brief history (Lecture 1) B. Basic concepts of Molecular Biology (Lecture 2-8) 1. Composition & structure of DNA & RNA, chromosomes, & genes 2. Genetic code 3. Replication, transcription, & translation C. Basic molecular tools & hybridization techniques (Lecture 9-14) 1. Restriction enzymes 2. Electrophoresis 3. Probes for direct detection of target DNA or RNA D. Amplification methods (Lecture 15-17) 1. Target Polymerase chain reaction (PCR) 2. Signal and Probe E. Human identity testing (DNA fingerprinting) (Lecture 18-26) ( 2
3 I. Molecular Diagnostics: Detection of specific DNA or RNA molecules II. Applications A. Infectious Diseases B. Genetic Diseases C.Oncology (Cancer) D.Histocompatability testing (HLA) E. Human Identity Testing (DNA Fingerprinting) 1. Forensics 2. Paternity ( 3
4 I s A. Gregor Mendel 1. Studied inheritance of pea plants 2. Discovered the basic laws of inheritance a. Inheritance of each trait determined by units or factors (now known as genes) a. Passed to descendents unchanged b. Individual inherits one such factor from each parent ( 4
5 I s B. Frederick Miescher 1. Took pus cells from discarded bandages 2. Isolated Nuclein, he correctly assumed from cell nuclei 3. Nuclein composition a. Acidic: Today know as deoxyribonucleic acid (DNA) b. Basic: Proteins 4. Question: Which portion responsible for cell inheritance? ( 5
6 II. Frederick Griffith - Transformation experiment (1928) A. Pneumococci 1. S strain: encapsulated; highly pathogenic 2. R strain: unencapsulated; nonpathogenic B. The experiment 1. S strain + mice dead mice 2. R strain + mice alive mice 3. Heat-killed S strain + mice alive mice 4. Mix of heat-killed S & live R strain + mice dead mice 5. Blood culture from dead mice S strain C. Conclusion: Something transformed the R strain to the S strain ( 6
7 I s A. Oswald Avery, Colin MacLeod & Maclyn McCarty 1. Determined transforming substance: Deoxyribonucleic acid (DNA) 2. Many scientists still thought protein coded for genetic material a. DNA too simple b. Protein more likely because more complex 3. Results confirmed when transforming factor a. Destroyed by deoxyribonucleases (DNAse) b. Unaffected by proteolytic enzymes ( 7
8 B. DNA composition determined 1. Four nitrogenous bases a. Purines i. Adenine (A) ii. Guanine (G) b. Pyrimidines i. Thymine (T) ii. Cytosine (C) 2. Deoxyribose (pentose sugar) 3. Phosphate ( 8
9 C. Erwin Chargaff s Rules 1. Base composition of DNA varies with species 2. DNA from different tissues from same species = same 3. DNA bases do not change with age, nutrition, environment 4. In all DNAs, A = T; G = C ( 9
10 III s A. X-ray diffraction studies (Rosalind Franklin & Maurice Wilkins) B. James Watson & Francis Crick 1. Proposed 3D model (1953) 2. Awarded Nobel Prize (along with Wilkins) 3. Key aspects of model a. Double-stranded (2 DNA strands) i. Antiparallel ii. 5,3 -phosphodiester bonds run in opposite directions b. Double helix (right-handed) i. Major groove ii. Minor groove c. Outside of helix: Hydrophilic backbone of deoxyribose & phosphate d. Purine and pyrimidine bases stacked internally i. Adenine paired with Thymine ii. Guanine paired with Cytosine e. DNA strands not identical but complementary ( 10
11 1. Define the term Molecular Diagnositics. 2. For each of the following briefly summarize his or her contribution to the understanding of Molecular Biology: a. Gregor Mendel b. Frederick Miescher c. Frederick Griffith d. Oswald Avery, Colin MacLeod & Maclyn McCarty e. Erwin Chargaff f. Rosalind Franklin & Maurice Wilkins g. James Watson & Francis Crick ( 11
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