17 sequencing

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17 sequencing

  1. 1. Fredrick Sanger Developed dideoxy termination sequencing method Nobel Laureate, 1980 Nobel Laureate, 1958; determined insulin sequence
  2. 2. DNA Sequencing The process is based on how DNA is replicated. The sequence of bases could be determined if: 1. DNA polymerase was stopped at each base 2. the last nucleotide added could be identified (AGCT)
  3. 3. DNA Sequencing The components of a sequencing reaction are: 1.DNA polymerase 2.DNA template strand 3.primer 4.deoxyribonucleotides (dNTPs) 5.dideoxyribonucleotides (ddNTPs)
  4. 4. dideoxyribose when both oxygen atoms removed 3’ OH functional group necessary for DNA elongation deoxyribose when oxygen removed Without the 3’ OH group, the DNA chain cannot be elongated from the dideoxyribose
  5. 5. DNA Sequencing Four reactions are set up; one for each possible nucleotide. A G T C 15% dATP 25% dATP 25% dATP 25% dATP 25% dGTP 15% dGTP 25% dGTP 25% dGTP 25% dTTP 25% dTTP 15% dTTP 25% dTTP 25% dCTP 25% dCTP 25% dCTP 15% dCTP 10% ddATP 10% ddGTP 10% ddTTP 10% ddCTP
  6. 6. DNA Sequencing components in one test tube
  7. 7. DNA Sequencing A G T C 15% dATP 25% dATP 25% dATP 25% dATP 25% dGTP 15% dGTP 25% dGTP 25% dGTP 25% dTTP 25% dTTP 15% dTTP 25% dTTP 25% dCTP 25% dCTP 25% dCTP 15% dCTP 10% ddATP 10% ddGTP 10% ddTTP 10% ddCTP
  8. 8. DNA Sequencing DNA Sequencing Video
  9. 9. DNA Sequencing Contents of each tube is separated by gel electrophoresis Which type of gel medium is best suited for this method? polyacrylamide
  10. 10. DNA Sequencing
  11. 11. DNA Sequencing
  12. 12. DNA Sequencing

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