Your SlideShare is downloading. ×
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
12 molecular techniques in  radiobiology
Upcoming SlideShare
Loading in...5
×

Thanks for flagging this SlideShare!

Oops! An error has occurred.

×
Saving this for later? Get the SlideShare app to save on your phone or tablet. Read anywhere, anytime – even offline.
Text the download link to your phone
Standard text messaging rates apply

12 molecular techniques in radiobiology

533

Published on

Published in: Technology
0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total Views
533
On Slideshare
0
From Embeds
0
Number of Embeds
0
Actions
Shares
0
Downloads
20
Comments
0
Likes
0
Embeds 0
No embeds

Report content
Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
No notes for slide

Transcript

  • 1. Molecular Techniques in Radiobiology佛教慈濟綜合醫院 放射腫瘤科 劉岱瑋
  • 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. 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. The Structure of DNA
  • 5. The Structure of DNA
  • 6. Hydrogen Bonds between Base Pairs
  • 7. The Structures of Nucleotide in DNA and RNA
  • 8. Central Dogma of Molecular Biology Exon Intron Exon Intron Exon DNAReplication Transcription Post-transcriptional modification mRNA Translation Post-translational modification Protein
  • 9. The Genetic Code
  • 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. 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. 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. VECTORS Plasmids Bacteriophage λ Cosmids Yeast artificial chromosome (YAC) Viruses
  • 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. Mammalian Plasmids
  • 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. 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. Bacteriophage λ
  • 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. Cosmids
  • 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. Yeast Artificial Chromosome (YAC)
  • 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. 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. 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. 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. 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. 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. Viral Vector as Gene Transfer Tools  Retrovirus  Adenovirus  Adeno-associated virus  Herpes simplex virus  Vaccinia virus  Sindbis virus
  • 30. Agarose Gel Electrophoresis
  • 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. 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. Functional Complementation
  • 34. Hybridization
  • 35. Oligonucleotide Probes
  • 36. Antibody Probes
  • 37. Positional Cloning
  • 38. Gene Analyses MappingSouthern Blotting
  • 39. Chromosome Walking
  • 40. DNA Sequence Analyses Chain-termination Method
  • 41. Restriction Fragment Length Polymorphism
  • 42. RestrictionFragment Length Polymorphism (RFLP)
  • 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. Comparisons between Southern,Northern and Western Blotting
  • 45. Study of Promoters:The CAT Assay
  • 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.

×