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Fig. 20-11

Northern Blot (to detect CD4 mRNA). TECHNIQUE. Heavy weight. I II III. RNA. Nitrocellulose membrane (blot). Gel. Sponge. T cells. B cells. macrophage. Paper towels. Fig. 20-11. Alkaline solution. 2. 1. 3. Preparation of restriction fragments. DNA transfer (blotting).

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Fig. 20-11

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  1. Northern Blot (to detect CD4 mRNA) TECHNIQUE Heavyweight I II III RNA Nitrocellulosemembrane (blot) Gel Sponge T cells B cells macrophage Papertowels Fig. 20-11 Alkalinesolution 2 1 3 Preparation of restriction fragments DNA transfer (blotting) Gel electrophoresis Radioactively labeledprobe forCD4 gene Probe base-pairswithmRNA I II III I II III Film overblot Nitrocellulose blot 4 5 Probe detection Hybridization with radioactive probe

  2. Reverse Transcriptase PCR (RT-PCR) to detect CD4 mRNA TECHNIQUE 1 cDNA synthesis mRNAs cDNAs Fig. 20-13 Primers 2 PCR amplification CD4 mRNA 3 Gel electrophoresis Different cell types RESULTS 1 2 3 4 5 6

  3. Microarrays to detect many (or all) mRNAs at once TECHNIQUE Tissue sample 1 Isolate mRNA. Fig. 20-15 2 Make cDNA by reversetranscription, usingfluorescently labelednucleotides. mRNA molecules Labeled cDNA molecules(single strands) DNA fragmentsrepresentingspecific genes 3 Apply the cDNA mixture to amicroarray, a different gene ineach spot. The cDNA hybridizeswith any complementary DNA onthe microarray. DNA microarray DNA microarraywith 2,400human genes 4 Rinse off excess cDNA; scanmicroarray for fluorescence.Each fluorescent spot represents agene expressed in the tissue sample.

  4. Example of array data genes WT dif1 ∆ dif1 myb98 ∆ myb98

  5. Table 21-1

  6. Human Genome Project (Multinational Consortium) 1990-2003 “Divide and conquer” approach Entire 3 x 10^9 nucleotide sequence of a composite haploid human genome ~$500 million - $1 billion Celera Genomics (Private Company) 1998-2003 Shotgun sequencing approach ~ $300 million

  7. Fig. 20-12a How can we sequence DNA? (Sanger dideoxy method) TECHNIQUE DNA(template strand) Primer Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged) dATP ddATP dCTP ddCTP dTTP ddTTP DNA polymerase dGTP ddGTP

  8. Fig. 20-12b TECHNIQUE DNA (template strand) Labeled strands Shortest Longest Directionof movementof strands Longest labeled strand Detector Laser Shortest labeled strand RESULTS Last baseof longestlabeledstrand Last baseof shortestlabeledstrand

  9. How can we sequence an entire genome? Linkage mapping 1 Genetic markers Physical mapping 2 Overlapping fragments DNA sequencing 3

  10. How can we sequence an entire genome? • Genome sequencing: • Divide and conquer approach • Ordered, large fragments of chromosomes are cloned Linkage mapping 1 Genetic markers Physical mapping 2 Overlapping fragments DNA sequencing 3

  11. Hummingbird cell TECHNIQUE Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Fig. 20-4-4 Nonrecombinant plasmid Recombinant plasmids Bacteria carryingplasmids RESULTS Colony carrying recombinant plasmid with disrupted lacZ gene Colony carrying non-recombinant plasmidwith intact lacZ gene One of manybacterial clones

  12. How can we sequence an entire genome? Genome sequencing: Divide and conquer approach - Ordered, large fragments of chromosomes are cloned -Each fragment is sequenced Linkage mapping 1 Genetic markers Physical mapping 2 Overlapping fragments DNA sequencing 3

  13. 1 Cut the DNA into overlapping fragments short enough for sequencing “Shotgun” sequencing approach 2 Clone the fragments in plasmid or phage vectors. 3 Sequence each fragment. 4 Order the sequences into one overall sequence with computer software.

  14. The Human Genome (partial sequence….) TTATTTCCCATTTTTCTTAAAAAGGAAGAACAAACTGTGCCCTAGGGTTTACTGTGTCAGAACAGAGTGTGCCGATTGTGGTCAGGACTCCATAGCATTTCACCATTGAGTTATTTCCGCCCCCTTACGTGTCTCTCTTCAGCGGTCTATTATCTCCAAGAGGGCATAAAACACTGAGTAAACAGCTCTTTTATATGTGTTTCCTGGATGAGCCTTCTTTTAATTAATTTTGTTAAGGGATTTCCTCTAGGGCCACTGCACGTCATGGGGAGTCACCCCCAGACACTCCCAATTGGCCCCTTGTCACCCAGGGGCACATTTCAGCTATTTGTAAAACCTGAAATCACTAGAAAGGAATGTCTAGTGACTTGTGGGGGCCAAGGCCCTTGTTATGGGGATGAAGGCTCTTAGGTGGTAGCCCTCCAAGAGAATAGATGGTGAATGTCTCTTTTCAGACATTAAAGGTGTCAGACTCTCAGTTAATCTCTCCTAGATCCAGGAAAGGCCTAGAAAAGGAAGGCCTGACTGCATTAATGGAGATTCTCTCCATGTGCAAAATTTCCTCCACAAAAGAAATCCTTGCAGGGCCATTTTAATGTGTTGGCCCTGTGACAGCCATTTCAAAATATGTCAAAAAATATATTTTGGAGTAAAATACTTTCATTTTCCTTCAGAGTCTGCTGTCGTATGATGCCATACCAGAGTCAGGTTGGAAAGTAAGCCACATTATACAGCGTTAACCTAAAAAAACAAAAAACTGTCTAACAAGATTTTATGGTTTATAGAGCATGATTCCCCGGACACATTAGATAGAAATCTGGGCAAGAGAAGAAAAAAAGGTCAGAGTTTAATCCTCATTCCTAAGTTATGTAAACCAAAAATAAAATTCTGAAGATGTCCTGATCATCTGAATGGACCCTTCCTCTGGACCAGGGCATTCCAAAGTTAACCTGAAAATTGGTTTGGGCCATGATGGGAAGGGAGGTTTGGATATGCCTCATTATGCCCTCTTCCCTTTCAGAATTCAGGAAAAGCCAACCAGCATTAACATCAACACAGATTTTCAGATCTTAGGTTTCTTTCCGATCTATTCTCTCTGAACCCTGCTACCTGGAGGCTTCATCTGCATAATAAAACTTTAGTCTCCACAACCCCTTATCTTACCCCAGACATTCCTTTCTATTGATAATAACTCTTTCAACCAATTGCCAATCAGGGTATGTTTAAATCTACCTATGACCTGGAAGCCCCCACTTTGCACCCTGAGATCAAACCAGTGCAAATCTTATATGTATTGATTTGTCAATGAAAACAGTCAAAGCCAGTCAGGCACAGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGCGGGTAGATCACCTGAGGTCAGGAGTTCGACACCAGCCTGGCCAACATGGTGAAACCCCGTCCCTACTAAAATACAAAAATTAGCCCAGCTTGGTGGTGGGCACCTGTAATCTTAGCTACTGCAGAGACTGAGGCAGGAGAATCGCTTGAACCCAGGAGGTGGAGGTTGCAGTGACCTGAGATTTTGCCATTGCACTCCAGCCTGGGCAACAGAGCAAGACTCTATCTCAAAAAACAAACAAACAAACAAACAAACAAACAAACTGTCAAAATCTGTACAGTATGTGAAGAGATTTGTTCTGAACCAAATATGAATGACCATGGTCCATGACACAGCCCTCAGAAGACCCTGAGAACATGTGCCCAAGGTGGTCACAGTGCATCTTAGTTTTGTACATTTTAGGGAGATATGAGACTTCAGTCAAATACATTTTTAAAAAATACATTGGTTTTGTCCAGAAAGCCAGAACCACTCAAAGCAGGGGTTTCCAGGTTATAAGTAGATTTAAAATTTTTCTGATTGACAATTGGTTGAAAGAGTTGTCAATAGAAAGGAATGTCTGCATTGTGACAAGAGGTTGTGGAGACCAAGTTTCTGTCATGCAGATGAAGCCTTCAGGTAGCAGGCTTCCAAGATAACAGGTTGTAAATAGTTCTTATCAGACTTAAGTTCTGTGGAGACGTAAAATGAGGCATATCTGACCTCCACTTCCAAAAACATCTGAGACAGGTCTCAGTTAATTAAGAAAGTTTGTTCTGCCTAGTTTAAGGACATGCCCATGACACTGCCTCAGGAGGTCCTGACAGCATGTGCCCAAGGTGGTCAGGATACAGCTTGCTTCTATATATTTTAGGGAGAAAATACATCAGCCTGTAAACAAAAAATTAAATTCTAAGGTCCCTGAACCATCTGAATGGGCTTTCTTCTAGGCCAGGGCACTCTAAAATTGAAGAACCTGAACATTCCTTTCTATTGATAATACTTTCAGCCAGTTGAGCCCATTCAGACCACAGCAAGGTGCCAGGCCAGGCAAGGGCTGACTTGAGATACCTGCCAGATGAGTCACTGGCAAAAGGTGCTGCTCCCTGGTGAGGGAGAAACACCAGGGGCTGGGAGAGGCCCAGAAGGCTCTGAAGGAGTTTTGGTTTGGCTGGCCATGTGTGCAATTAGCGTGATGAGCTCTGACATGGCCTTGCATGGACGGATTGGGCAGGACACCCCAGCTGAGGAGGATGGCAGGAGTGATGGCACAGGGGAAAGGGTGGCATACCCAGGTGACAGCTCCCCACTACCTCCACTCTGTGCTGCAGCTCAGGGGCTGGGTCTTCTGCTGCAACTCAGCCCCTCTGTACCAGCCCTGGCCTCATTCCCTTGGTTCCAGGACACCCAGCTGACAAAAGGGACTTGCCTGTACCCCTGCACCTGGTCCTACACCTGGCTCCTGGGTTGTCAGCAGGTGTTTGTTGGGCCAACGAGTGCATGGATGGAAACACAGACAGAAGGACAGATGGAGAGATGGTGGGTGGCCAGACAAAGGAGTAACTTGGTGAGGAATGTGCATTAGGAAATCACAGAAGAGCAGAAACTGTTTGAAAATTCCAAGTGGGGAAAGTGAGGAGGTGAAGCAGGGCTGAAGGGCCTCCCTCAGAGCCTTCTCCCACTCTGTGGTGTCCACATCCCCTTGGTCGTCCTTGTGGGAGGCACTCACCTTTTGCTCAGCCTATTGTGGCTACAGCCCAGCAGGTCCCAGGTGGCACCAGCCAAGATGAAGGTGGCATTGAGGGCTGAAGTCTCCCTCACCATGAAGGGATGATGTATAGTGGGTGGGGCCTCAGGAGGAAGAG GGCCACCAACCCTACCTGGCCCCTAACCTGCTGCCTGGAGTAGGCAGGTACCAGAGGCATGGGGTGAGGCATGTTGCAGGTCGAGGACCAGGGCCATCTCACTGCCTGAGCCCATGGACTGGCTCAGGGGTCTGTCAGATGATTCTAGAGCTGAGTTGGAGGTAAGGGCAGGGGGTTTGTTCCTGGGTTCAAGACCATGGAAGGAAGGGGTAGAGAAGGAGGCCAACAAGTGAGGAGGCAAATTACAGTGGCTGGCAGAAGGAGAGAGAAGCCAGGACAGGTGGCTGTGGCCCTGTCCCTGCAGGCAGACCCAGGAAGGAGCTCAGAGACAGGATTCATGCCAAGCCTGCCTACCCAGCACATCTCTCCTCATGGACATGAGAGAAAACCCTCCAGCTTGGCCCTCACATCTGTGAAACCCACAGTAATGGGGCTGACATCCTCTGCCCTATGCAAGAGAGGTTTCCCAAGCACTTGCAGCAAGTGAGACTGCACAGGATGGCGAATCCACAAAGAACACGTTGTTCTCATGCTCTTTGGAAGCACCAATTTACATTCTG

  15. Table 21-1

  16. Exons (regions of genes coding for protein or giving rise to rRNA or tRNA) (1.5%) Repetitive DNA that includes transposable elements and related sequences (44%) Introns and regulatory sequences (24%) Fig. 21-7 Unique noncoding DNA (15%) L1 sequences (17%) Repetitive DNA unrelated to transposable elements (15%) Alu elements (10%) Simple sequence DNA (3%) Large-segment duplications (5–6%)

  17. Comparison of gene organization in different species (centered on region containing RNA polymerase gene)

  18. New copy of transposon Transposon Fig. 21-9a DNA of genome Transposon is copied Insertion Mobile transposon (a) Transposon movement (“copy-and-paste” mechanism)

  19. New copy of retrotransposon Retrotransposon Fig. 21-9b RNA Insertion Reverse transcriptase (b) Retrotransposon movement

  20. Large scale sequencing of cDNA fragments Reverse Transcriptase PCR (RT-PCR) TECHNIQUE 1 cDNA synthesis mRNAs cDNAs Primers 2 PCR amplification Sequence large numbers (millions) of cDNA fragments 3 Gel electrophoresis

  21. Large scale sequencing of cDNA fragments No UV (3 samples) UV (3 samples) Fragments matching rad51

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