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Honors ~ Dna 1314
Honors ~ Dna 1314
Honors ~ Dna 1314
Honors ~ Dna 1314
Honors ~ Dna 1314
Honors ~ Dna 1314
Honors ~ Dna 1314
Honors ~ Dna 1314
Honors ~ Dna 1314
Honors ~ Dna 1314
Honors ~ Dna 1314
Honors ~ Dna 1314
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Honors ~ Dna 1314
Honors ~ Dna 1314
Honors ~ Dna 1314
Honors ~ Dna 1314
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Honors ~ Dna 1314

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  • 1. Molecular Biology Honors Biology Edgar
  • 2. Hershey and Chase 1952
  • 3. Agarose
  • 4. Separation of DNA fragments by Size
  • 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. 2652 2652
  • 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. DNA Replication
  • 9. Fig. 16-UN5
  • 10. Fig. 16-13 Topoisomerase Helicase PrimaseSingle-strand binding proteins RNA primer 5′ 5′ 5′ 3′ 3′ 3′
  • 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. Fig. 16-16a Overview Origin of replication Leading strand Leading strand Lagging strand Lagging strand Overall directions of replication 1 2
  • 13. Helicase
  • 14. Topoisomerase and Helicase
  • 15. Fig. 20-3-1 Restriction site DNA Sticky end Restriction enzyme cuts sugar-phosphate backbones. 5′ 3′ 3′ 5′ 1
  • 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. 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. 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. Fig. 20-9b RESULTS
  • 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. Transcription and Translation
  • 22. Beadle and Tatum 1941
  • 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. Gene Regulation
  • 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. Gene Regulation Example 1 Activators, Enhancers and Transcription Factors
  • 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. 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. 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. Fig. 18-9-1 Enhancer TATA box PromoterActivators DNA Gene Distal control element
  • 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. 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. 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. Gene Regulation Example 2 The Operon
  • 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. 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. Fig. 18-3b-1 (b) Tryptophan present, repressor active, operon off Tryptophan (corepressor) No RNA made Active repressor mRNA Protein DNA
  • 38. Fig. 18-3b-2 (b) Tryptophan present, repressor active, operon off Tryptophan (corepressor) No RNA made Active repressor mRNA Protein DNA
  • 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. 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. 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. Gene Regulation Example 3 Epigenetics
  • 43. Epigenetics
  • 44. Epigenetics Intro http://learn.genetics.utah.edu/content/epigenetics/intro/
  • 45. Utah Epigenetics http:// learn.genetics.utah.edu/content/epigenetics/intro/movies/epigenome
  • 46. Gene Regulation Example 4 RNAi
  • 47. RNAi
  • 48. RNA Induced Silencing Complex
  • 49. Vascular Endothelial Growth Factor
  • 50. Human Genome
  • 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. Transformation – Recombinant Organisms
  • 53. Cloning Technologies
  • 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. 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. 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. 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. DNA Laboratory at Milton Academy • Isolate DNA from cheek cells. • Polymerase Chair Reaction • Electrophoresis • Sequence DNA
  • 59. mtDNA Control Region
  • 60. Polymerase Chain Reaction
  • 61. PCR http://www.dnalc.org/resources/spotlight/index.html
  • 62. Taq DNA Polymerase
  • 63. Fig. 20-8a 5′ Genomic DNA TECHNIQUE Target sequence 3′ 3′ 5′
  • 64. Fig. 20-8b Cycle 1 yields 2 molecules Denaturation Annealing Extension Primers New nucleo- tides 3′ 5′ 3 2 5′ 3′1
  • 65. Fig. 20-8c Cycle 2 yields 4 molecules
  • 66. Fig. 20-8d Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence
  • 67. http://www.youtube.com/watch?v=CQEaX3MiDow http://www.youtube.com/watch?v=x5yPkxCLads&feature=related
  • 68. Gel Electrophoresis
  • 69. DNA Sequencing
  • 70. Fredrick Sanger
  • 71. Chain Termination Methods Sanger Methods
  • 72. Dye-terminator sequencing
  • 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. Fig. 20-12a DNA (template strand) TECHNIQUE DNA polymerase Primer Deoxyribonucleotides Dideoxyribonucleotides (fluorescently tagged) dATP dCTP dTTP dGTP ddATP ddCTP ddTTP ddGTP
  • 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. Trace File
  • 77. Amplification and clonal selection
  • 78. Kate Bator Connor Johnson
  • 79. High-throughput sequencing Next-Gen Sequencing
  • 80. mtDNA Sequence http://www.dnalc.org/view/15979-A-mitochondrial-DNA-sequence.html
  • 81. “The Other Genome” mtDNA
  • 82. Endosymbiotic Theory
  • 83. Mitochondrial Eve
  • 84. 100 Years 1 bp/sec 17 Minutes
  • 85. Human mtDNA Haplotypes
  • 86. mtDNA – Genographic Project Video
  • 87. Two Opposing Theories • Multiregional Theory – Parallel evolution • Displacement Theory – Out of Africa theory http://news.bbc.co.uk/
  • 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

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