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1.27.2010 lecture

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1.27.2010 lecture

  1. 1. 1.27.2010<br /><ul><li>Lecture Start
  2. 2. You must know nucleic acid or gene, you must know the sequence to make a single stranded probe.
  3. 3. Can do hybridization procedures with the single stranded DNA.
  4. 4. % formamide and temperature. 50% 37 degrees C may result in high pairing.
  5. 5. 50% 35 degress C may result in imperfect matching in double helix.
  6. 6. Can then pull out region of interst.
  7. 7. Can do same thing with specific sequences of RNA
  8. 8. Riboprobes - Produced by in vitro transcription.
  9. 9. Take sequence of DNA and insert into plasmid behind a promoter construct that is regulated by viral RNA polymerases.
  10. 10. Bacteria incorporate the plasmid and the viral bacteria will continue to transcribe the DNA into RNA.
  11. 11. Label the nucleotides so when the RNA is made, it’s labeled.
  12. 12. Dedoxygenin (marker) that is converted into a precipitate by enzymes so you can visualize in the cell
  13. 13. Radioactive, fluorescent, and enzymatic markers.
  14. 14. In this transcription process, you make sense and antisense molecules.
  15. 15. How are the DNA or RNA probes used?
  16. 16. To detect the DNA or RNA of interest in the cell.
  17. 17. Northern blots are used to identify RNA – don’t localize or tell where in the cell a protein is.
  18. 18. Same procedure as southern blot, except using different buffers, and lets you see RNA
  19. 19. Southern blots are used to identify DNA - don’t localize or tell where in the cell a protein is.
  20. 20. Take DNA in cell, denature, expose to radioactive probes that attach to DNA that has been transferred onto filter. Probes only target the sequences you are interested in. Rinse excess radioactivity/fluorescence.
  21. 21. In Situ hybridization
  22. 22. Probe is localized in the cell to a specific site and then you can view the genes or sequences while they are still in the living cell/chromosome.
  23. 23. FISH
  24. 24. (fluorescence in situ hybridization) is a cytogenetic technique used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence similarity. Fluorescence microscopy can be used to find out where the fluorescent probe bound to the chromosomes are. FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification. FISH can also be used to detect and localize specific mRNAs within tissue samples. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues.
  25. 25. Genes localized by specific probes
  26. 26. Can see in a chromosome
  27. 27. Can label RNA in a cell
  28. 28. How do you amplify samples if your proteins are scarce?
  29. 29. PCR
  30. 30. Usually only 1 sample
  31. 31. RT-PCR
  32. 32. More than 1 sample
  33. 33. Identify the presence of nucleic acids in low concentrations.
  34. 34. Reverse transcriptase polymerase chain reaction
  35. 35. Start with message from sample.
  36. 36. Collect all mRNA
  37. 37. Hybridize with a poly-t-primer at end of sample that will reverse transcribe cDNA
  38. 38. Uses single stranded mRNA as template
  39. 39. Degrade RNA strand with tail and left with cDNA
  40. 40. Use polymerase to amplify cDNA sequence
  41. 41. Now you have a double cDNA copy.
  42. 42. Amplify
  43. 43. Separate cDNA using high temps (95) and cool down (50) to add primers, which gives the DNA polymerase something to hang on to.
  44. 44. DNA polymerase is heat stable, but will only work at about 70 degrees C.
  45. 45. Add nucleotides to sample so the DNA polymerase can use to make cDNA strand.
  46. 46. Tac polymerase – needs Mg2+, nucleotides, single stranded template, double stranded region of DNA.
  47. 47. Amplification of about 10^9 times.
  48. 48. Must know what sequences you’re looking for…
  49. 49. Micro-arrays
  50. 50. Whether genes are upregulated or down regulated in certain conditions.
  51. 51. Takes advantages of chips that are generated where you have multiple single stranded copies of genes with themes (growth factors, cytokines etc)
  52. 52. Take samples and isolate mRNA then convert to cDNA and label with fluorescent probe. Hybridize to microarray. Where are the green/red/yellow spots? The key for the chips tells which single stranded DNA that is or which gene is that that has been bound to a cDNA construct from sample XYZ. Targets differences between two samples or in certain conditions.
  53. 53. Techniques used to alter protein expression
  54. 54. What is the role of a protein in cell?
  55. 55. Overproduce a protein to determine its function
  56. 56. Use DNA plasmid vectors
  57. 57. Promoter sequence
  58. 58. Cut plasmid
  59. 59. Insert particular DNA sequence behind promoter
  60. 60. Transform bacteria
  61. 61. Plasmid can be expressed and make protein of interest
  62. 62. Protein function can be dependent on concentration, so be careful.
  63. 63. Will misinterpret if there is a negative outcome
  64. 64. In order to determine if protein has been expressed, cut, antibodies on western blot to determine level of expression, correlate back to determine changes in cell
  65. 65. Or … Identify protein of interest specifically by tagging
  66. 66. Insert gene of interest into a construct that has a particular tag, such as histidine tags with Ni -HA tags – Histidine w/ nickel
  67. 67. M2 tags – protein structure is recognized by flag antibody
  68. 68. GST tag – glutiphinyl s transferase tag, can also be identified by antibodies for GST
  69. 69. Look for tag itself on western blot
  70. 70. Antibodies – tag so you don’t need to generate an expesivie antibody for protein
  71. 71. Purifies protein rapidly, tags separate on column.
  72. 72. Make sure tag isn’t doing anything to cell function
  73. 73. How to use proteins, that are themselves fluorescent, to identify proteins of interests
  74. 74. GFP is a tag that has a construct that has an inducible promoter and has several sites for restriction endonucleases to insert DNA of interest. The construct won’t be expressed until you add the sugar that activates it. ITPG sugar activates lac promoter and the lac promoter begins to turn on transcription and translation of the protein plus GFP.
  75. 75. You can use living cells with GFP and fluorescent microscopy.
  76. 76. We can insert a gene of interest and we have a gene that confers drug resistance to those cells that are transfected with the plasmid. Not all cells stably transfect into genome. Those that have, will have ampicilin resistance. After a while, only those that have stably incorporated live. All others die off.
  77. 77. Maybe you wanna eliminate protein function.
  78. 78. Replacement – difficult in mammalian cells
  79. 79. Knockout to remove active gene – difficult in mammalian, so you use embryonic stem cells injected into target gene replaced by mutation to generate progeny in a pregnant mouse that now expresses mutant/altered gene. Some have the mutation. Mate mice with normal mice so in F2, look for males and females with copies of mutated genes. Can be in somatic cells. Transgenic mice are looked at for the function of that gene.
  80. 80. Add a competing gene (gene addition)
  81. 81. RNAi
  82. 82. Knock-down experiment
  83. 83. Double stranded RNA has been known to be considered viral.
  84. 84. Double stranded RNA will be targeted by dicer, which cleaves the ends of the double stranded RNA making an siRNA (small interference RNA). The presence of siRNA is an indicator of these mechanisms of infection.
  85. 85. RISC proteins attach to siRNAs and degrade 1 strand of siRNA leaving a single strand which can:
  86. 86. Bind to message that has been generated which inhibits translation
  87. 87. Enter nucleus and bind complementary DNA to inhibit transcription.

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