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17 sequencing-2010 update
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17 sequencing-2010 update

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Transcript

  • 1.  
  • 2. Fredrick Sanger
    • developed dideoxy termination sequencing method
    • Nobel Laureate, 1980
    • Nobel Laureate, 1958; determined insulin sequence
  • 3. DNA Sequencing
    • similar to DNA replication
    • somehow stop at each base
    • determine which was the last base added
  • 4. Sequencing Requirements
    • DNA polymerase
    • DNA template strand
    • primer (radioactive)
    • deoxyribonucleotides
    • dideoxyribonucleotides
  • 5. deoxyribose when oxygen removed dideoxyribose when both oxygen atoms removed 3’ OH functional group necessary for DNA elongation
  • 6. Sequencing Setup
    • one test tube for each dideoxyribonucleotide (ddATP, ddGTP, ddTTP, ddCTP)
    A G T C 15% ATP 25% ATP 25% ATP 25% ATP 25% GTP 15% GTP 25% GTP 25% GTP 25% TTP 25% TTP 15% TTP 25% TTP 25% CTP 25% CTP 25% CTP 15% CTP 10% ddATP 10% ddGTP 10% ddTTP 10% ddCTP
  • 7. Sequencing Setup
    • one test tube for each dideoxyribonucleotide (ddATP, ddGTP, ddTTP, ddCTP)
    A G T C 15% ATP 25% ATP 25% ATP 25% ATP 25% GTP 15% GTP 25% GTP 25% GTP 25% TTP 25% TTP 15% TTP 25% TTP 25% CTP 25% CTP 25% CTP 15% CTP 10% ddATP 10% ddGTP 10% ddTTP 10% ddCTP
  • 8. Sequencing Setup components in one test tube
  • 9. After Replication Process
    • contents of each tube is separated by gel electrophoresis
    • Look at your notes.
  • 10.  
  • 11. SANGER SEQUENCING http://www.dnalc.org/view/15479-Sanger-method-of-DNA-sequencing-3D-animation-with-narration.html Insulin http://www.dnalc.org/view/16515-Animation-23-A-gene-is-a-discrete-sequence-of-DNA-nucleotides-.html