12 molecular techniques in radiobiology

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12 molecular techniques in radiobiology

  1. 1. Molecular Techniques in Radiobiology佛教慈濟綜合醫院 放射腫瘤科 劉岱瑋
  2. 2. Historical Perspectives Fred Griffith (1928) – Toxicity of Streptococcus Oswald Avery (1943) – DNA as the transforming factor Erwin Chargaff (1950) – Discover one-to-one ratio between adenine and thymine; cytosine and guanine Linus Pauling (1951) – Precise measurement of helical polypeptide structure James Watson and Francis Crick (1953) – Describe the structure of DNA Stanley Cohen (1972) – First recombinant DNA molecules, pSC101
  3. 3. The Structure of DNA Two antiparallel helices Deoxyribose sugar, Phosphate group, Nucleotide base Complementary relationship between base pairs 5’ to 3’ direction sequence DNA self-replication during cell division
  4. 4. The Structure of DNA
  5. 5. The Structure of DNA
  6. 6. Hydrogen Bonds between Base Pairs
  7. 7. The Structures of Nucleotide in DNA and RNA
  8. 8. Central Dogma of Molecular Biology Exon Intron Exon Intron Exon DNAReplication Transcription Post-transcriptional modification mRNA Translation Post-translational modification Protein
  9. 9. The Genetic Code
  10. 10. Restriction Endonuclease Type II Enzyme Type III Enzyme Type I EnzymeProtein structure Separate Bifunctional enzyme of Bifunctional endonuclease and 2 subunits enzyme of 3 methylase subunitsRecognition site 4-6 bp sequence, 5-7 bp Bipartite and often palindromic Asymmetric sequence asymmetricCleavage site Same as or close to 24-26 bp downstream Nonspecific > 1000 recognition site of recognition site bp from recognition siteRestriction & Separate reactions Simultaneous Nutually exclusivemethylation
  11. 11. Restriction EndonucleaseEnzyme Element of Terminology MeaningHindIII H Genus Haemophilus in Species influenzae d Strain Rd III Third endonuclease isolatedEcoRI E Genus Escherichia co Species coli R Strain RY13 I First endonuclease isolatedBamHI B Genus Bacillus am Species amylolquefaciensi H Strain H I First endonuclease isolated
  12. 12. Restriction EndonucleaseEndonuclease DNA Sequence Cleavage Products GGATCC G GATCC “Sticky” BamH I ends CCTAGG CCTAG G GAATTC G AATTC “Sticky” EcoR I ends CTTAAG CTTAA G AAGCTT A AGCTT “Sticky” Hind III ends TTCGAA TTCGA A GG CC GG CC Blunt Hae II CC GG CC GG ends CTGCAG CTGCA G “Sticky” Pst I GACGTC G ACGTC ends CCC GGG CCC GGG Blunt Sma I GGG CCC GGG CCC ends
  13. 13. VECTORS Plasmids Bacteriophage λ Cosmids Yeast artificial chromosome (YAC) Viruses
  14. 14. Plasmids Are found in a wide variety of bacterial species Are extrachromosomal elements that behave as accessary genetic units Have involved a variety of mechanisms to maintain a stable copy number of the plasmid in their bacterial hosts Are dependent on the enzymes and proteins encoded by their host for their replication and transcription Frequently contain genes coding for enzymes that are advantageous to the bacterial host
  15. 15. Mammalian Plasmids
  16. 16. Bacteriophage λ Bacteriopahge λ was first used as a cloning vector in the early 1970s The genome of bacteriophage λ is a double-stranded DNA molecules, 48502 bp in length The DNA is carried in bacteriophage particles as a linear double-straded molecules with single-straded termini 12 nucleotides in length (cohensive termini or cos) After entering a host bacterium, the cohensive termini associate by base pairing to form a circular molecules, then recombines into the E. coli chromosome
  17. 17. Bacteriophage λ Mixtures of extracts prepared from bacteria infected with stains of bacteriophage λ carrying mutations in genes required for the assembly of bacteriophage particles – In vitro packaging (1975) Bacteriophage λ can infect its host at a much higher efficiency than a plasmid Bacteriophage λ can accommodates DNA inserts up to about 24,000 bp
  18. 18. Bacteriophage λ
  19. 19. Cosmids Cosmid vectors are conventional plasmids that contain one or two copies of a small region of bacteriophage λ DNA – cohensive end site (cos) The cos contains all of the cis-acting elements required for packaging of viral DNA into bacteriophage λ particles Cosmids contain an antibiotic resistance gene to allow selection of infected cells
  20. 20. Cosmids
  21. 21. Yeast Artificial Chromosome (YAC) YAC are linear DNA molecules whose architecture mimics that of authentic yeast chromosome YAC contains a centromere, telomeres and selectable markers Most YAC libraries contain 250 kb and 400 kb of foreign DNA per clone
  22. 22. Yeast Artificial Chromosome (YAC)
  23. 23. Viral Vectors SV40 was the first viral vector for introducing foreign genes into mammalian cells Retrovirus are ideal vectors for introducing genes into mammalian cells in a stable fasion Adenoviral vectors Adeno-associated viral vectors Lentiviral vectors
  24. 24. Genomic Library Extraction of genomic DNA from tissue or culture cells Partial digestion by restriction endonuclease, EcoRI Genomic DNA fragments about 40,000 bp in size are ligated into a cosmic vector and packaged inside becteriophage particles The assembled bacteriophage particles are infected E. coli cells and selected by appropriate antibiotics
  25. 25. cDNA Library cDNA is DNA that is complementary to the mRNA and therefore includes only the expressed genes of a particular cell Extraction of total mRNA from tissue or culture cells Reverse transcription to form cDNA fragments and ligated to plasmids or bacteriophage λ Expression library can be screened using an antibody
  26. 26. Host Escherichia coli – Most widely used organism in molecular biology Yeast – simple eukaryotes but grow as quickly and inexpensively as bacteria, yeast mutants can serve as screening method in radiobiology Mammalian cells – - Short-term explants of cells, primary culture - Established cell line
  27. 27. Transformation• Alteration in cell growth pattern Increased growth to higher cell density Increase rate of growth Decrease requirement for serum growth factor Anchorage-independent growth Loss of contact inhibition -- foci• Alterations in cell surface Increase rate of transport of cell nutrients Increase secretion of protease or protease activator Increase agglutinability of glycoproteins and glycolipids Change in composition of glycoproteins, glycolipids• Alterations in intracellular components and biochemical process Increase metabolic rate Increase glycosis Altered levels of cyclicnucleotides Activation or repression of certain cellular genes Change in cell cytoskeleton -- round• Tumorgenesis in nude mice
  28. 28. DNA-mediated Gene Transfer Microinjection Calcium phosphate precipitation - The most widely used method of gene transfer in vitro - The efficiency varies markedly from one cell line to another Liposome vector -- Complex of cationic lipid and DNA Electroporation Gene gun Viral vectors
  29. 29. Viral Vector as Gene Transfer Tools  Retrovirus  Adenovirus  Adeno-associated virus  Herpes simplex virus  Vaccinia virus  Sindbis virus
  30. 30. Agarose Gel Electrophoresis
  31. 31. Polymerase Chain Reaction (PCR) Enzymatic amplification of DNA fragment Forward and reward primers Heat-stable Taq DNA polymerase DNA strand denature at 94 ℃ Primers anneal to template at specific temperature DNA elongate at 72 ℃
  32. 32. Gene-Cloning Strategies Choose a source of DNA - Genomic DNA or cDNA Construction a genomic or cDNA library Screen the library to locate the gene
  33. 33. Functional Complementation
  34. 34. Hybridization
  35. 35. Oligonucleotide Probes
  36. 36. Antibody Probes
  37. 37. Positional Cloning
  38. 38. Gene Analyses MappingSouthern Blotting
  39. 39. Chromosome Walking
  40. 40. DNA Sequence Analyses Chain-termination Method
  41. 41. Restriction Fragment Length Polymorphism
  42. 42. RestrictionFragment Length Polymorphism (RFLP)
  43. 43. Single-stranded Conformation Polymorphism (SSCP) Rely on the differences in mobility between single- stranded DNA molecules on the basis of their secondary structures in nondenaturing gels DNA molecules of identical length but different secondary structure will migrate at different rates in nondenaturing electrophoretic gels DNA fragments can be isolated or synthesized by performing PCR on patient DNA samples, they can then be denatured, and individual strands allowed to reanneal to themselves SSCP can only detect about 80% of such mutations
  44. 44. Comparisons between Southern,Northern and Western Blotting
  45. 45. Study of Promoters:The CAT Assay
  46. 46. microRNA & siRNA in mammalian microRNA: endogenous (nucleus) siRNA:exogenous (cytoplasm)MicroRNAs: small RNAs with a big role in gene regulation.He L, Hannon GJ.

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