Genetics in the News Recombinant DNA Technology

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Genetics in the News Recombinant DNA Technology

  1. 1. Genetics in the News
  2. 2. Recombinant DNA Technology <ul><li>…artificial manipulation of DNA. </li></ul>
  3. 3. <ul><ul><li>isolate DNA, </li></ul></ul><ul><ul><li>cut DNA into workable sized fragments, </li></ul></ul><ul><ul><li>amplify the fragments for storage and subsequent analysis, </li></ul></ul><ul><ul><li>identify and isolate specific sequences, </li></ul></ul><ul><ul><li>characterize by size genomic location and sequence. </li></ul></ul>Recombinant DNA Technology
  4. 4. Cutting the DNA <ul><li>~3,100,000,000 base pairs per human haploid genome, </li></ul><ul><ul><li>average chromosome length is ~13 million base pairs, </li></ul></ul><ul><li>DNA is cut with bacteria-derived enzymes, </li></ul><ul><li>The act of cutting is termed “ digestion ”. </li></ul>
  5. 5. Restriction Enzymes <ul><li>…proteins that recognize specific, short nucleotide sequences and cut DNA at those sites, </li></ul><ul><li>…bacteria contain over 400 such enzymes and recognize and cut over 100 DNA sequences. </li></ul>
  6. 6. Bacteria Defense Mechanism <ul><li>restriction enzymes are expressed in bacteria in order to protect against invasion by viruses, </li></ul><ul><li>bacterial protect their own DNA by chemically modifying recognition sites. </li></ul>
  7. 7. Palindromes <ul><li>stir grits </li></ul><ul><li>no lemon, no melon </li></ul>5 ’ -------G-A-A-T-T-C--------3 ’ 3 ’ -------C-T-T-A-A-G--------5 ’
  8. 8. EcoRI 5 ’ -------G A-A-T-T-C--------3 ’ 3 ’ -------C-T-T-A-A G--------5 ’
  9. 9. Sticky Ends <ul><li>...single-stranded DNA overhangs resulting from restriction digestion. </li></ul>
  10. 10. EcoRI 5 ’ -------G A-A-T-T-C--------3 ’ 3 ’ -------C-T-T-A-A G--------5 ’
  11. 11. RsaI Rhodopseuomonas sphaeroides 5 ’ -------G-T A-C--------3 ’ 3 ’ -------C-A T-G--------5 ’
  12. 13. Restriction Fragment Length Sizes (predicted) <ul><li>- IF - </li></ul><ul><li>25% A 25% T 25% G 25% C </li></ul><ul><li>and </li></ul><ul><li>Random Distribution of Nucleotides </li></ul>- THEN - Distance between cut sites is equal to 4 n bases , (n = number of base pairs in the recognition site) i.e. p(specific base) = .25
  13. 14. Average Restriction Fragment Length <ul><ul><li>n = 4, 256 base pairs </li></ul></ul><ul><ul><li>n = 6, 4096 base pairs </li></ul></ul><ul><ul><li>n = 8, 65.5 kb base pairs </li></ul></ul>
  14. 16. Still Lots of Fragments <ul><li>4 cutter: 3,000,000,000 bp </li></ul><ul><li>~256 bp = 12,000,000 fragments </li></ul><ul><li>6 cutter: 3,000,000,000 bp </li></ul><ul><li>~4096 bp = 700,000 fragments </li></ul><ul><li>8 cutter: 3,000,000,000 bp </li></ul><ul><li>~65.5 kb = 46,000 fragments </li></ul>
  15. 17. Ligation <ul><li>… sticky ends with complementary base pairs can form hydrogen bonds, </li></ul><ul><li>… DNA ligase: an enzyme that catalyzes the reformation of the phosphodiester bonds. </li></ul>
  16. 18. Ligation 5 ’ -------G A-A-T-T-C--------3 ’ 3 ’ -------C-T-T-A-A G--------5 ’ hydrogen bonds align, DNA ligase covalently links
  17. 19. DNA is Profligate <ul><li>…sticky ends with complementary base pairs can form hydrogen bonds, </li></ul><ul><li>…with DNA from any species. </li></ul>
  18. 20. Cloning … specialized DNA technology to produce multiple, exact copies of a single gene or other segment of DNA to obtain enough material for further study.
  19. 21. <ul><li>Requires a Vector… </li></ul><ul><li>...a specialized DNA sequence that can enter a living cell, </li></ul><ul><li>...signal its presence to an investigator, </li></ul><ul><li>... and provide a means of replication for itself and the foreign DNA it carries. </li></ul>Cloning … specialized DNA technology to produce multiple, exact copies of a single gene or other segment of DNA to obtain enough material for further study.
  20. 22. And, Vectors… <ul><li>… contain unique restriction sites to facilitate the creation of recombinant DNA molecules, </li></ul><ul><li>... must also possess a distinguishing physical characteristic such as size or shape by which it can be purified away from the host cells genome. </li></ul>Mike
  21. 23. Vectors <ul><li>Plasmid E. coli up to 15 kb, </li></ul><ul><li>Phage E. coli up to 25 kb, </li></ul><ul><li>Cosmid E. coli up to 45 kb, </li></ul><ul><li>BAC E. coli 100-500 kb, </li></ul><ul><li>YAC Yeast 250-1000 kb. </li></ul>
  22. 24. Step 1…restriction digests and ligation of fragments into cloning vectors,
  23. 25. Cloning Step 2 <ul><li>… vector-insert recombinants are inserted in host cells, </li></ul><ul><ul><li>Transformation (via bacterial mechanisms); </li></ul></ul><ul><ul><ul><li>plasmids </li></ul></ul></ul><ul><ul><ul><li>BACS </li></ul></ul></ul><ul><ul><ul><li>YACS </li></ul></ul></ul><ul><ul><li>Transduction (via virus mechanisms); </li></ul></ul><ul><ul><ul><li>phage </li></ul></ul></ul><ul><ul><ul><li>cosmids </li></ul></ul></ul>
  24. 26. Cloning Step 3 <ul><li>...vectors contain selectable markers, </li></ul><ul><ul><li>only cells that contain vector DNA will survive selection, </li></ul></ul><ul><ul><li>recombinant vectors can be discerned from empty vectors by additional markers. </li></ul></ul>Blue/White Cloning
  25. 27. Amplification <ul><li>…if you were to grind me up with a giant mortar and pestle, and extract all of my DNA coding for a single gene, you’d get about 1  g of DNA, </li></ul><ul><li>…two liters of bacterial culture carrying my hemoglobin in a recombinant DNA vector, would yield an equivalent mass of DNA. </li></ul>
  26. 28. Assignment <ul><li>Read 10.1 and be prepared to clone a fragment of DNA using a plasmid as a vector. </li></ul>
  27. 29. B-PCR vs A-PCR <ul><li>PCR, P olymeras C hain R eaction </li></ul>
  28. 30. Today More DNA Science <ul><li>DNA Amplification II, </li></ul><ul><ul><li>Polymerase Chain Reaction , </li></ul></ul><ul><ul><li>6.7, pp. 237-239, </li></ul></ul><ul><li>Gel Electrophoresis, </li></ul><ul><ul><li>Southern Blots, Northern Blots, </li></ul></ul><ul><ul><li>6.6, pp. 237-238, </li></ul></ul><ul><li>Clones and Libraries, pp </li></ul>
  29. 31. P olymerase C hain R eaction <ul><li>… invented by Kary Mullis while cruising in a Honda Civic on Highway 128 from San Francisco to Mendocino, </li></ul><ul><li>&quot;It was quiet and something just went, Click!&quot; </li></ul>Kary B. Mullis Nobel Laureate, 1993 Chemistry
  30. 32. <ul><li>&quot; THE SUN HAD been hot that day in Mendocino County. A dry wind had come out of the east, and nobody knew how hot it had been until, around sunset, the wind stopped. I drove up from Berkeley through Cloverdale headed to Anderson Valley. The California buckeyes poked heavy blossoms out into Highway 128. The pink and white stalks hanging down into my headlights looked cold, but they were loaded with warmed oils that dominated the dimension of smell. </li></ul><ul><li>It seemed to be the night of the buckeyes, but something else was stirring.&quot;&quot;My little silver Honda's front tires pulled us through the mountains. My hands felt the road and the turns. My mind drifted back to the lab. DNA chains coiled and floated. Lurid blue and pink images of electric molecules injected themselves somewhere between the mountain road and my eyes.&quot; </li></ul>Opening words, Dancing Naked in the Mind Field , © 1998, by Dr. Kary Mullis , Pantheon Books.
  31. 33. Mullis… <ul><li>... “PCR is a chemical procedure that will make the structures of the molecules of our genes as easy to see as billboards in the desert and as easy to manipulate as Tinkertoys”. </li></ul>DNA
  32. 34. Making DNA: Components test tube nucleus Environment ? DNA polIII DNA Polymerase ? primase Primer present present dNTPs ? helicase, etc. ss DNA template PCR Cell
  33. 35. Oligonucleotides specific primers <ul><li>...short pieces of synthetic DNA can be manufactured that contain any sequence, </li></ul><ul><li>… template specific! </li></ul>~ Odds of a Specific Sequence 20-mer: 9.1 x 10 -13
  34. 36. Making One Strand Of DNA Add Polymerase Add dNTPs, etc. add primer
  35. 37. Making Two More Strands Must Denature Separate Strands
  36. 38. Denaturing can’t use helicase in vitro <ul><li>… DNA denaturing conditions such as high heat or low salt concentrations irreversibly denature or inactivate most polymerases, </li></ul><ul><li>… dNTPs are not affected by denaturation, </li></ul><ul><li>… primers are not affected by denaturation. </li></ul>
  37. 39. Making Two More Strands Add polymerase, etc. add primer to second strand
  38. 40. Denaturation Step Bad <ul><li>…several rounds of in vitro replication could be performed (prior to PCR), however, accumulation of denatured polymerases quickly poisoned the reactions. </li></ul>
  39. 41. <ul><li>… bacteria discovered in a hot spring in Yellowstone Natural Park in 1965, </li></ul><ul><li>… lives in salty water that ranges from 70 o - 75 o C, </li></ul><ul><li>… thus, does DNA replication at very high temperatures. </li></ul>
  40. 42. Thermus aquaticus’ Enzymes <ul><li>… basic research demonstrated that many enzymes isolated from Thermus aquaticus function at very high temperatures, </li></ul><ul><li>… temperatures nearing 100 o C, </li></ul><ul><li>… DNA denaturating temperatures. </li></ul>
  41. 43. Click <ul><li>… Kary Mullis realized that repetitive rounds of DNA synthesis could be performed by using a heat-stable polymerase, </li></ul><ul><li>… T hermus aq uaticus : Taq polymerase. </li></ul>
  42. 44. 94 o ~60 o 72 o Synthesis ~30 seconds/kb PCR 3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’ 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’ 5’--GCATGCATTAT CTGATCGTGAC--5’ Denature Step ~30 seconds 3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’ 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’ 5’--GCATGCATTAT CTGATCGTGAC--5’ Annealing Step ~30 seconds 3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’ 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’ 5’--GCATGCATTAT CTGATCGTGAC--5’ 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’ 3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’ 5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’ 3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’
  43. 45. Exponential Synthesis <ul><li>as few as 1 DNA templates required, </li></ul><ul><li>excess dNTPS, </li></ul><ul><li>excess primers, </li></ul><ul><li>multiple cycles. </li></ul>
  44. 46. Gel Electrophoresis <ul><li>… electrophoresis is the movement of charged particles in an electric field, </li></ul><ul><li>… DNA with it’s phosphate backbone carries a net negative charge, </li></ul><ul><ul><li>DNA migrates toward the positive pole in an electrical field. </li></ul></ul>
  45. 47. Size Resolution <ul><li>… DNA movement during electophoresis is dependent on several variables, </li></ul><ul><ul><li>strength of the electrical field, </li></ul></ul><ul><ul><li>composition of the matrix (gel), </li></ul></ul><ul><ul><li>charge per unit volume of the molecule, </li></ul></ul><ul><ul><ul><li>each nucleotide has roughly equal charge, </li></ul></ul></ul><ul><ul><ul><li>all DNA molecules have the same charge density, </li></ul></ul></ul><ul><ul><li>size. </li></ul></ul>
  46. 48. Agarose Gel Agarose: a natural polysaccharide derived from agar, a substance found in some algae.
  47. 49. Add DNA to Wells, Apply Charge DNA moves through gel at a rate roughly equivalent to the inverse log of the number of base pairs.
  48. 50. Visualizing DNA Ethidium Bromide fluoresces in UV light.
  49. 51. Looks Like This
  50. 52. PCR Applications <ul><li>…new applications are created every day, </li></ul><ul><li>PCR products can be used for mapping genes, </li></ul><ul><li>PCR products can be used as probes, </li></ul><ul><li>PCR products can be probed, </li></ul><ul><li>PCR can be used to identify genotypes, </li></ul><ul><li>PCR can be used to sequence DNA directly. </li></ul>
  51. 53. Genetics … in the news. … a hardcopy of the assigned paper will earn you two of the 12.5 points on the quiz.
  52. 54. Syllabus Update <ul><li>6.1, 6.6 - 6.8, 10.1 for Wednesday, </li></ul><ul><ul><li>read the rest of the chapter for review, </li></ul></ul><ul><ul><li>Master the Chapter 6 summary, </li></ul></ul><ul><ul><li>Master 6.8, 6.13, 6.18, 6.19, 10.6, 10.7, 10.9, 10.11, </li></ul></ul><ul><ul><li>Quiz through Chapter 6, 7.1 - 7.5, 10.1 Wednesday. </li></ul></ul><ul><li>Read the Science Paper by Friday. </li></ul>
  53. 56. Molecular Probing Heterologous Hybridization <ul><li>… genes (DNA), or gene products (RNA) can be identified based on hybridization to labeled molecules, </li></ul><ul><li>… DNA probes are short, single-stranded stretches of nucleic acid that are complementary to target nucleic acids, </li></ul><ul><ul><li>10 - 1000s of base pairs in length, </li></ul></ul><ul><li>… radioactive or fluorescent labeled for detection. </li></ul>
  54. 57. Probe Add a “labelled” dNTP to an in vitro synthesis reaction.
  55. 58. Southern Blot
  56. 59. Northern Blot mRNA is... RNA
  57. 60. DNA Libraries <ul><li>… collections of cloned DNA fragments, </li></ul><ul><ul><li>genomic, </li></ul></ul><ul><ul><li>cDNA (coding sequences). </li></ul></ul>
  58. 61. Genomic Sequences and Coverage <ul><li>N = ln(1 - P ) </li></ul><ul><li>ln(1 - f ) </li></ul><ul><li>N = number of clones </li></ul><ul><li>P = probability of recovering a sequence, </li></ul><ul><li>f = fraction of the genome of each clone </li></ul>
  59. 62. Genomic Sequences and Coverage <ul><li>N = ln(1 - .99) </li></ul><ul><li>ln(1 - v /2,900,000,000) </li></ul><ul><li>v = average vector insert size </li></ul><ul><li>plasmid (5000 bp) = 2.7 x 10 6 </li></ul><ul><li>phage (20 kb) = 6.7 x 10 5 </li></ul><ul><li>BAC (125 kb) = 1.0 x 10 5 </li></ul><ul><li>YAC (500 kb) = 27,000 clones </li></ul>
  60. 63. E. coli vs. Humans P = probability of including any one sequence. v/g = insert size / genome size E. coli Genome = 4.6 mb n = 4.6 mb / 20 kb insert = 230 P = 0.999 # Clones = 1585 Human Genome = 2900 mb n = 2900 mb / 20 kb insert = 145,000 P = 0.999 # Clones = 1,001,621 # Clones = ln(1 - P) ln(1 - v/g )
  61. 65. cDNA <ul><li>…DNA synthesized from an mRNA template with the enzyme reverse transcriptase . </li></ul>
  62. 66. Reverse Transcriptase <ul><li>1. RNA dependent, DNA synthesis. </li></ul><ul><li>2. RNA Degradation. </li></ul><ul><li>3. DNA dependent, DNA Synthesis. </li></ul>Error Rate: 1 in 20,000 nucleotides.
  63. 67. mRNA -AAAA... - polyadenylation. - introns are spliced out.
  64. 68. cDNA Construction in vitro
  65. 69. cDNA ?
  66. 70. Genomic vs cDNA <ul><li>...Jeff’s hemoglobin… </li></ul><ul><li>isolate red blood cells, </li></ul><ul><ul><li>red blood cells make lots of hemoglobin, thus the mRNA is enriched for hemoglobin sequences, </li></ul></ul><ul><li>construct a cDNA library, </li></ul><ul><li>isolation of hemoglobin clones is facilitated, </li></ul><ul><ul><li>genomic: ~1 of 1,000,000 clones, </li></ul></ul><ul><ul><li>cDNA from red blood cells: 1 of 3 clones. </li></ul></ul>
  67. 71. cDNA Libraries <ul><li>…provide a ‘snap-shot’ of the genes expressed in a particular cell, at a particular time, or under specific condition, </li></ul><ul><li>…however, do not provide regulatory sequences. </li></ul>
  68. 73. Assignment <ul><li>Study figure 6.27, pp 237, </li></ul><ul><li>Be able to describe the steps required to isolate a genomic clone using a cDNA clone as a molecular probe. </li></ul>
  69. 74. Primers?
  70. 77. Cycle Sequencing PCR: 1 Strand Sequencing <ul><li>PCR driven DNA sequence procedure, </li></ul><ul><ul><li>non-exponential amplification, </li></ul></ul><ul><li>Dideoxy sequencing method, </li></ul><ul><ul><li>florescent indicators. </li></ul></ul>
  71. 78. Single Strand PCR Template dNTPs 1 Primer Taq Polymerase w/ Buffer Cycles = Polymerization until Taq falls off, linear amplification.
  72. 79. Cycle Sequencing Chain Termination Template ddNTPs 1 Primer Taq Polymerase w/ Buffer Cycles = Polymerization until Taq hits ddNTP, Taq falls off. dNTPs
  73. 80. Fluorescent ddNTPs Plus: a preponderance of dNTPs
  74. 82. Cycle Sequencing Chain Termination Template ddNTPs dNTPs Lots of each sized fragment are produced, each with a specific florescent base on the end.base etc. Another Template
  75. 83. For Friday! … READ IT! BRING YOUR COPY!
  76. 84. Exam #1 <ul><li>Increase your study time, or see me to work on study approaches. </li></ul>Mean = 72% Median = 72% Mode = 76% A range: | | | | | | | B range: | | | | | | | C range: | | | | | | | | | | | | D range: | | | | | | | | | | | Failing: | | | | | | | | | | | | |
  77. 85. Assignment: figure out this pedigree. 14/50 Albinism is a recessive trait in humans.

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