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Lecture 16

Lecture 16. Nucleic Acid Chemistry. Hydrolysis of RNA. Occurs under alkaline conditions. Hydrolysis of RNA. Nucleic Acid Chemistry. Denaturation (increase T). RNA-RNA>RNA-DNA RNA-DNA>DNA-DNA. Nucleotides Absorb UV Light. Base stacking decreases absorption Hypochromic effect.

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Lecture 16

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  1. Lecture 16 Nucleic Acid Chemistry

  2. Hydrolysis of RNA • Occurs under alkaline conditions

  3. Hydrolysis of RNA

  4. Nucleic Acid Chemistry Denaturation (increase T) RNA-RNA>RNA-DNA RNA-DNA>DNA-DNA

  5. Nucleotides Absorb UV Light • Base stacking decreases absorption • Hypochromic effect

  6. Denaturation

  7. Deamination

  8. Alkylation

  9. Depurination

  10. Pyrimidine Dimers

  11. DNA photolyase Native DNA uv light Pyrimidine dimer DNA photolyase binds ( Light (>300nm) Light used to correct dimer Enzyme releases repaired DNA

  12. DNA Sequencing • Mid 1970s • Alan Maxam and Walter Gilbert • Chemical cleavage method • Fred Sanger • Chain-terminator method • Dideoxynucleotides

  13. Maxam Gilbert DNA Sequencing 1. Cleave DNA strand at specific nucleotides -e.g. A only 2. Separate fragments according to size by electrophoresis

  14. Sanger Method • Simpler and more widely used • Uses DNA replication by DNA polymerase • All DNA polymerases • Require primer + template • Synthesize 53

  15. DNA Primer and Template

  16. ddNTP Analog

  17. Sanger Method

  18. Sanger Method

  19. Sanger Method Gel

  20. Automation of DNA sequencing

  21. Automation of DNA Sequencing

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