Honors ~ Dna 1314

1,017 views

Published on

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

No Downloads
Views
Total views
1,017
On SlideShare
0
From Embeds
0
Number of Embeds
625
Actions
Shares
0
Downloads
9
Comments
0
Likes
1
Embeds 0
No embeds

No notes for slide

Honors ~ Dna 1314

  1. 1. Molecular Biology Honors Biology Edgar
  2. 2. Hershey and Chase 1952
  3. 3. Agarose
  4. 4. Separation of DNA fragments by Size
  5. 5. Looking at your gels • What do you notice about the “banding patterns” in each lane in your gels? • What is different about the “pools” of DNA that you loaded into each well?
  6. 6. 2652 2652
  7. 7. Look at you gel again • Estimate the size of the DNA fragment(s) in the pMAP lane. • Does the relationship between the distance migrated and DNA fragment size appear to be a linear relationship?
  8. 8. DNA Replication
  9. 9. Fig. 16-UN5
  10. 10. Fig. 16-13 Topoisomerase Helicase PrimaseSingle-strand binding proteins RNA primer 5′ 5′ 5′ 3′ 3′ 3′
  11. 11. Fig. 16-16b6 Template strand 5′ 5′3′ 3′ RNA primer 3′ 5′ 5′ 3′ 1 1 3′ 3′ 5′ 5′ Okazaki fragment 12 3′ 3′ 5′ 5′ 12 3′ 3′ 5′ 5′ 1 2 5′ 5′ 3′ 3′ Overall direction of replication
  12. 12. Fig. 16-16a Overview Origin of replication Leading strand Leading strand Lagging strand Lagging strand Overall directions of replication 1 2
  13. 13. Helicase
  14. 14. Topoisomerase and Helicase
  15. 15. Fig. 20-3-1 Restriction site DNA Sticky end Restriction enzyme cuts sugar-phosphate backbones. 5′ 3′ 3′ 5′ 1
  16. 16. Fig. 20-3-2 Restriction site DNA Sticky end Restriction enzyme cuts sugar-phosphate backbones. 5′ 3′ 3′ 5′ 1 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. 2 One possible combination
  17. 17. Fig. 20-3-3 Restriction site DNA Sticky end Restriction enzyme cuts sugar-phosphate backbones. 5′ 3′ 3′ 5′ 1 One possible combination Recombinant DNA molecule DNA ligase seals strands. 3 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. 2
  18. 18. Fig. 20-9a Mixture of DNA mol- ecules of different sizes Power source Longer molecules Shorter molecules Gel AnodeCathode TECHNIQUE 1 2 Power source – + +–
  19. 19. Fig. 20-9b RESULTS
  20. 20. Fig. 20-10 Normal allele Sickle-cell allele Large fragment (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles 201 bp 175 bp 376 bp (a) DdeI restriction sites in normal and sickle-cell alleles of β-globin gene Normal β-globin allele Sickle-cell mutant β-globin allele DdeI Large fragment Large fragment 376 bp 201 bp175 bp DdeIDdeI DdeI DdeI DdeI DdeI
  21. 21. Transcription and Translation
  22. 22. Beadle and Tatum 1941
  23. 23. Development of Model • One Gene – One Enzyme (Nobel 1958) • One Gene – One Polypeptide – Non enzyme proteins (keratin, insulin) – Hb – multimeric protein. • Issues: – Alternate splicing – RNA coding genes. – Non-coding regions
  24. 24. Gene Regulation
  25. 25. Fig. 18-6 DNA Signal Gene NUCLEUS Chromatin modification Chromatin Gene available for transcription Exon Intron Tail RNA Cap RNA processing Primary transcript mRNA in nucleus Transport to cytoplasm mRNA in cytoplasm Translation CYTOPLASM Degradation of mRNA Protein processing Polypeptide Active protein Cellular function Transport to cellular destination Degradation of protein Transcription
  26. 26. Gene Regulation Example 1 Activators, Enhancers and Transcription Factors
  27. 27. Fig. 18-8-1 Enhancer (distal control elements) Proximal control elements Poly-A signal sequence Termination region Downstream Promoter Upstream DNA ExonExon ExonIntron Intron
  28. 28. Fig. 18-8-2 Enhancer (distal control elements) Proximal control elements Poly-A signal sequence Termination region Downstream Promoter Upstream DNA Exon Exon ExonIntronIntron Cleaved 3′ end of primary transcript Primary RNA transcript Poly-A signal Transcription 5′ ExonExon ExonIntron Intron
  29. 29. Fig. 18-8-3 Enhancer (distal control elements) Proximal control elements Poly-A signal sequence Termination region Downstream Promoter Upstream DNA ExonExon ExonIntron Intron Exon Exon ExonIntronIntron Cleaved 3′ end of primary transcript Primary RNA transcript Poly-A signal Transcription 5′ RNA processing Intron RNA Coding segment mRNA 5′ Cap 5′ UTR Start codon Stop codon 3′ UTR Poly-A tail 3′
  30. 30. Fig. 18-9-1 Enhancer TATA box PromoterActivators DNA Gene Distal control element
  31. 31. Fig. 18-9-2 Enhancer TATA box PromoterActivators DNA Gene Distal control element Group of mediator proteins DNA-bending protein General transcription factors
  32. 32. Fig. 18-9-3 Enhancer TATA box PromoterActivators DNA Gene Distal control element Group of mediator proteins DNA-bending protein General transcription factors RNA polymerase II RNA polymerase II Transcription initiation complex RNA synthesis
  33. 33. Fig. 18-10 Control elements Enhancer Available activators Albumin gene (b) Lens cell Crystallin gene expressed Available activators LENS CELL NUCLEUS LIVER CELL NUCLEUS Crystallin gene Promoter (a) Liver cell Crystallin gene not expressed Albumin gene expressed Albumin gene not expressed
  34. 34. Gene Regulation Example 2 The Operon
  35. 35. Fig. 18-2 Regulation of gene expression trpE gene trpD gene trpC gene trpB gene trpA gene (b) Regulation of enzyme production (a) Regulation of enzyme activity Enzyme 1 Enzyme 2 Enzyme 3 Tryptophan Precursor Feedback inhibition
  36. 36. Fig. 18-3a Polypeptide subunits that make up enzymes for tryptophan synthesis (a) Tryptophan absent, repressor inactive, operon on DNA mRNA 5′ Protein Inactive repressor RNA polymerase Regulatory gene Promoter Promoter trp operon Genes of operon Operator Stop codonStart codon mRNA trpA 5′ 3′ trpR trpE trpD trpC trpB ABCDE
  37. 37. Fig. 18-3b-1 (b) Tryptophan present, repressor active, operon off Tryptophan (corepressor) No RNA made Active repressor mRNA Protein DNA
  38. 38. Fig. 18-3b-2 (b) Tryptophan present, repressor active, operon off Tryptophan (corepressor) No RNA made Active repressor mRNA Protein DNA
  39. 39. Fig. 18-4a (a) Lactose absent, repressor active, operon off DNA Protein Active repressor RNA polymerase Regulatory gene Promoter Operato r mRNA 5′ 3′ No RNA made lacI lacZ
  40. 40. Fig. 18-4b (b) Lactose present, repressor inactive, operon on mRNA Protein DNA mRNA 5′ Inactive repressor Allolactose (inducer) 5′ 3′ RNA polymerase Permease Transacetylase lac operon β- Galactosidase lacYlacZ lacAlacI
  41. 41. Fig. 18-5 (b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized cAMP DNA Inactive lac repressor Allolactose Inactive CAP lacI CAP-binding site Promoter Active CAP Operator lacZ RNA polymerase binds and transcribes Inactive lac repressor lacZ OperatorPromoter DNA CAP-binding site lacI RNA polymerase less likely to bind Inactive CAP (a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized
  42. 42. Gene Regulation Example 3 Epigenetics
  43. 43. Epigenetics
  44. 44. Epigenetics Intro http://learn.genetics.utah.edu/content/epigenetics/intro/
  45. 45. Utah Epigenetics http:// learn.genetics.utah.edu/content/epigenetics/intro/movies/epigenome
  46. 46. Gene Regulation Example 4 RNAi
  47. 47. RNAi
  48. 48. RNA Induced Silencing Complex
  49. 49. Vascular Endothelial Growth Factor
  50. 50. Human Genome
  51. 51. Encode The Encyclopedia of DNA Elements http://www.youtube.com/watch?v=TwXXgEz9o4w&feature=player_d http://www.youtube.com/watch?v=Y3V2thsJ1Wc&feature=player_de
  52. 52. Transformation – Recombinant Organisms
  53. 53. Cloning Technologies
  54. 54. Fig. 20-4-1 Bacterial cell Bacterial plasmid lacZ gene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends ampR gene TECHNIQUE
  55. 55. Fig. 20-4-2 Bacterial cell Bacterial plasmid lacZ gene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends ampR gene TECHNIQUE Recombinant plasmids Nonrecombinant plasmid
  56. 56. Fig. 20-4-3 Bacterial cell Bacterial plasmid lacZ gene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends ampR gene TECHNIQUE Recombinant plasmids Nonrecombinant plasmid Bacteria carrying plasmids
  57. 57. Fig. 20-4-4 Bacterial cell Bacterial plasmid lacZ gene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends ampR gene TECHNIQUE Recombinant plasmids Nonrecombinant plasmid Bacteria carrying plasmids RESULTS Colony carrying non- recombinant plasmid with intact lacZ gene One of many bacterial clones Colony carrying recombinant plasmid with disrupted lacZ gene
  58. 58. DNA Laboratory at Milton Academy • Isolate DNA from cheek cells. • Polymerase Chair Reaction • Electrophoresis • Sequence DNA
  59. 59. mtDNA Control Region
  60. 60. Polymerase Chain Reaction
  61. 61. PCR http://www.dnalc.org/resources/spotlight/index.html
  62. 62. Taq DNA Polymerase
  63. 63. Fig. 20-8a 5′ Genomic DNA TECHNIQUE Target sequence 3′ 3′ 5′
  64. 64. Fig. 20-8b Cycle 1 yields 2 molecules Denaturation Annealing Extension Primers New nucleo- tides 3′ 5′ 3 2 5′ 3′1
  65. 65. Fig. 20-8c Cycle 2 yields 4 molecules
  66. 66. Fig. 20-8d Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence
  67. 67. http://www.youtube.com/watch?v=CQEaX3MiDow http://www.youtube.com/watch?v=x5yPkxCLads&feature=related
  68. 68. Gel Electrophoresis
  69. 69. DNA Sequencing
  70. 70. Fredrick Sanger
  71. 71. Chain Termination Methods Sanger Methods
  72. 72. Dye-terminator sequencing
  73. 73. Fig. 20-12 DNA (template strand) TECHNIQUE RESULTS DNA (template strand) DNA polymerase Primer Deoxyribonucleotides Shortest Dideoxyribonucleotides (fluorescently tagged) Labeled strands Longest Shortest labeled strand Longest labeled strand Laser Direction of movement of strands Detector Last base of longest labeled strand Last base of shortest labeled strand dATP dCTP dTTP dGTP ddATP ddCTP ddTTP ddGTP
  74. 74. Fig. 20-12a DNA (template strand) TECHNIQUE DNA polymerase Primer Deoxyribonucleotides Dideoxyribonucleotides (fluorescently tagged) dATP dCTP dTTP dGTP ddATP ddCTP ddTTP ddGTP
  75. 75. Fig. 20-12b TECHNIQUE RESULTS DNA (template strand) Shortest Labeled strands Longest Shortest labeled strand Longest labeled strand Laser Direction of movement of strands Detector Last base of longest labeled strand Last base of shortest labeled strand
  76. 76. Trace File
  77. 77. Amplification and clonal selection
  78. 78. Kate Bator Connor Johnson
  79. 79. High-throughput sequencing Next-Gen Sequencing
  80. 80. mtDNA Sequence http://www.dnalc.org/view/15979-A-mitochondrial-DNA-sequence.html
  81. 81. “The Other Genome” mtDNA
  82. 82. Endosymbiotic Theory
  83. 83. Mitochondrial Eve
  84. 84. 100 Years 1 bp/sec 17 Minutes
  85. 85. Human mtDNA Haplotypes
  86. 86. mtDNA – Genographic Project Video
  87. 87. Two Opposing Theories • Multiregional Theory – Parallel evolution • Displacement Theory – Out of Africa theory http://news.bbc.co.uk/
  88. 88. Neandertal Genome Study Reveals That We Have a Little Caveman in Us Svante Paabo Europeans and Asians share 1% to 4% of their nuclear DNA with Neandertals. But Africans do not

×