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2011-11-17 13:41:39

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  1. in gel electrophoresis
    DNA fragment are separated by size as they move through a gel matrix. it is one of the most useful means of separating and purifying DNA fragments for further analysis
  2. migration of the DNA
    • the blue dye is negatively charged and migrate tawords the positive electrode
    • the smallest fragment moves fastest being entangled less in the agarose matrix of the gel
  3. fluorescence dye
    used to stain the DNA fragment
  4. production of labeled DNA probes
    • 2 methods
    • biotinylated or fluorescin-congugated nucleotides into the cDNA
    • labelling is performed by the incorporation of 32p-labelled nucleotides
  5. to detect specific DNA sequences
    only one practical way to fine one particular DNA segment is to exploit its sequence specificity
  6. production of labled DNA probes:
    2 methods
    • biotinylated or fluorescin-conjugated nucleotides into the cDNA
    • labelling is performed by the incoporation of 32p-lablelled nucleotieds
  7. southern bloting
    • fragmentation of DNA by digestion with ristriction endonucleases
    • fragments are separated by gel electrophoresis.
    • the gel is soaked in a solution of NAOH. alkali denatures duplex DNA, converting it to single-strand DNA.
    • neutrtralize with buffer
    • the bans are transferred to nitrocellulose membrane using southern blot technique nitrocellulose binds single stranded DNA molecules very tightly, immobilizing them on the sheet.
    • incubation with DNA labeled probe at low stringencies, hybridization with many DNA , mebrane is then washed at high stringency, probe remains bound only to high homologous sequences.
    • detection by autoradiographic exposuer of a piece of X-ay film laid over the nitrocellulose sheet.
  8. northen blotting:???
    western blotting:??
    • northen blotting is for mRNA
    • western blotting is for proteins
  9. rDNA
    DNA molecule constructed with DNA from different sources
  10. rDNA technology referes to techniques that are used to:
    • manipulate, move, recombine and propergate DNA
    • produce a desired protein in large quantities.
  11. princible of rDNA technology
    • foregin DNA sequences can be inserted into plasmid vectors by opening the circular plasmid with the restriction endonucleae
    • the ends of the linearized plasmid DNA are then joined with the endes of foreign sequence, reclosing the cirle to create a chimerical plasmid.
  12. prokaryotic cells
    • advantages:
    • 1-express greater concentration of protein product(compared to eukaryotic cells)
    • 2-require relatively simple media componeents
    • limitations:
    • 1-do not perform many important post translational modifications( ex: glycosylation).
    • 2- it is not possible to express large proteins in E.coli.
  13. endonucleases
    restriction enzymes
    • recognize specific base sequences in dsDNA
    • break the phosphodiester bonds between 2 nucleotides within the sequence
  14. endonucleases
    sticky end:???
    • some enzymes cut the DNA at a different position in the 2 strands,producing a single stranded overhang or a sticky end ex: Eco RI
    • other enzyme produce blunt-ended fragments with no sticky end. ex: PvuII
  15. sticky end endonucleases
    the formation of sticky ends is useful by base pair matching of the sticky ends, any DNA fragment produced by the action of the same restriction enzyme can be joined together in a specific manner.
  16. 2 DNA ligases are used in DNA analysis:
    • DNA ligase from E.coli which requires the nucleotide NAD+ as a cofactor
    • T4 DNA ligase from T4 phage which requires ATP as a cofactor.
  17. DNA ligases
    • catalalyze the synthesis of a phospho-diester bond between:
    • 1- the 3' -OH group of 1 nucleotide
    • 2- the 5' phoshporyl group of the next nucleotide in a DNA molecule.
    • ligases are most often used to produce a covalent bond between nucleotides in DNA joined by base pair matching of sticky end
  18. primers:???
    primes are short oligonucleotieds complementary to the ends of the original DNA or RNA strand, are added to the incubatiion mixture.
  19. DNA polymerase I
    • it attaches to short single standed region in a dsDNA molecule
    • then it synthesizes a new strand of DNA, degrading the existing strand as it proceeds.
    • in vitro, it must be incubated at 12-15 C to prevent more than one round of replication occuring
    • the DNA polymerase I molecule contains its polymerase and nuclease activity on different parts of the enzyme molecule.
  20. klenow fragment
    • contain the part which retains the polymerase function
    • it synthesize a new DNA strand complementary to the single strand of DNA( the template) only.
    • it is used to create blunt ends.
  21. reverse transcriptase
    it synthesize a cDNA strand using mRNA as a template
  22. taq polymerase
    • it has its maximum activity at 70-80 C
    • it remains active up to 90 C
    • it is used in PCR
  23. DNA modifying enzymes
    • enzyme that can be used to remove or add groups to the ends of the DNA molecule:
    • 1- alkaline phosphatase which removes a phospate group from the 5'terminus
    • 2- polynucleotide kinase which adds a phosphate group to a free 5' terminus
    • 3- terminal deoxynucleotidyl transferase which add 1 or more deoxynucleotides to the 3' terminus
  24. methods for DNA modification
    • plasmid
    • bacteriophages
  25. plasmid:??
    • is a circular DNA duplex
    • an extrachromosomal segment of DNA found in certain bacteria
    • is replicated by the cell
    • contains the requisite gentic machinery, such as a replication origin, to permit their autonomous propagation in a bacterial host or yeast.
  26. why plasmids are good cloning vectors
    • 1- small size (1 to 200 Kb) thus easy to manipulate and isolate
    • 2- circular ( more stable than linear DNA)
    • 3- replication is independent of the host cell
    • 4- several copies may be present ( facilitates replication)
    • 5- frequently have antibody resistance ( detection easy)
  27. steps of rDNA technology or cloning steps
    • 1- identification of the protein to be produced
    • 2- isolation of the gene of interest
    • 3-insertion into a suitable vector (plasmid)
    • 4-insertion of theplasmid into host cell
    • 5-culture ----> clones
    • 6-detection and purification of the desired clone. replicate clone to high numbers
  28. bacteriophages
    • 1- are viruses that infect bacteria used as vectors
    • 2- linear dsDNA containg genes are required for viral replication within the host cell.
    • 3- during infection DNA molecules are inserted into the host bacteria
    • 4- 2 modes of replication:
    • A- with cell lysis
    • B- without cell lysis
  29. steps of DNA modification using phage:
    • 1-isolation of DNA from phage and digestion with restriction enzymes
    • 2- connection of fragments to foreign DNA using DNA ligase
    • 3- packing of DNA with head and tail proteins
    • 4- infection of bacterial host
    • 5- detection of transferred genes
  30. issues with gene therapy
    • 1-we could genetically engineer any "desirable" trait, but ethically it is un unacceptable
    • 2- we cannot deliver rDNA to human eggs or sperms in order to ensure that all cells have the gene.
    • 3- it can only be done with cells that can be removed form the body, engineered and cultured in vitro and then returend to the body ex: white blood cells. that's why it is called "EX vivo" gene therapy
    • 4- gene therapy of some genetic disease require the delivery of rDNA to different cell types, most of which cannot be easily removed and reintroduced---> will require new technological advances. ex: good cycstic fibrosis gen is introduced via a recombinant adenovirus cia an aerosal spray.
  31. how do you know wheter a disorder is a good candidate for gene therapy?
    • 1- does the condition result form mutation in one or more genes?
    • 2- which genes are involved
    • 3- what do you know about the biology of the disorder
    • 4- will adding a normal copy of the gene fix the problem in the affected tissue
    • 5- can you deliver the gene to cells of the affected tissue.
  32. gene delivery: the key to gene therapy
    • 1- targeting the right cells
    • 2- activating the gene
    • 3- integrating the gene in the cells
    • 4- avoiding harmful side effects
  33. vectors
    • adinovirus
    • retrovirus
    • adeno-associated virus
    • herps simplex virus
    • liposome
    • naked DNA
  34. dominant negative
    • when adding a good copy of the gene won't solve the problem.
    • deal with dominant negative through:
    • 1- a technique for reparing mutations: SMaRT( spliceosome-mediated RNA trans-splicing)
    • 2- techniques to prevent the production of a muatated protein:
    • A- triplex-helix forming oligonucleotides
    • B- antisense
    • c- ribozyme
  35. retrovirus
    • RNA ex: HIV
    • affect only dividing cells.
    • 8 kbp
    • converted to DNA before activating
    • integrate randomly
    • S.E: disturb other genes because of intigration randomly
  36. adenoviruse
    • ds DNA: ex common cold
    • dividing and non-dividing
    • 7.5kbp
    • it will not integrate, gene activation lost after week or 2
    • S.E: imune response
  37. adino associated viruse
    • ssDNA
    • dividing and non dividing
    • need helper viruse to replicate themselves inside cells
    • 5 kbp
    • 95% integrated at a specific region on chromosome 19 greatly reducing the chance that integration will disrupt the fn of other genes in the cells
    • NO imune response
  38. herpes simplix virus
    • ssDNA ex: oral and genetl herps
    • infect cells of nervous system
    • max 20kbp
    • don't integrate but remain for long time as a separate circular piece of DNA that replicate when cell divide and it will not disturbe the function of other genes
    • SE: imune response
  39. liposomes
    • circular dsDNA called plasmid
    • not viral type
    • no specific target, less effective, no max lenght
    • will not integrate unless engeneerd
    • don't induce immune response but some time are toxic
    • better suited for ex vivo gene therapy approach
  40. naked DNA
    • circular dsDNA called plasmid
    • not viral type
    • no specific target, less effective, no max lenght
    • will not integrate unless engeneerd
    • don't induce immune respon and non toxic
    • better suited for ex vivo gene therapy approach