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DNA Replication and PCR

DNA Replication and PCR. Central Dogma. DNA structure. nucleotides. Pentose (5 Carbon) sugar. phosphate. At physiological pH phosphate group is charged. ?? What implications does this have for DNA molecules?. Bases. purines. pyrimidines. How are DNA monomers put together.

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DNA Replication and PCR

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  1. DNA Replication and PCR

  2. Central Dogma

  3. DNA structure

  4. nucleotides

  5. Pentose (5 Carbon) sugar

  6. phosphate At physiological pH phosphate group is charged ?? What implications does this have for DNA molecules?

  7. Bases purines pyrimidines

  8. How are DNA monomers put together Phosphodiester bond what type of bond is this? covalent

  9. What holds helices together ? Hydrogen bonds

  10. What has fallen apart? + = DNA + Boiling water = Denaturation hydrogen bonding is disrupted

  11. ConceptsWhat do you need to know? • How does DNA polymerase work • Concept of molecular machines

  12. How does DNA polymerase work? • DNA polymerase can only extend pre-existing molecules • DNA polymerase can move only in direction: 5’3’

  13. How does DNA polymerase work?

  14. Will this system make DNA? dATP dGTP dCTP dTTP

  15. Will this system make DNA? dATP dGTP dCTP dTTP

  16. Will this system make DNA? dATP dGTP dCTP dTTP

  17. Will this system make DNA?yes dATP dGTP dCTP dTTP

  18. DNA polymerase can move only in direction: 5’3’ dATP dGTP dCTP dTTP

  19. DNA polymerase can move only in direction: 5’3’ 5’ 3’ 5’ 3’ dATP dGTP dCTP dTTP

  20. Molecular Machinery

  21. dNTPS vs NTPs

  22. Inventor Kary B. Mullis Nobel in Chemistry in 1993 Process for Amplifying Nucleic Acid Sequences Polymerase Chain Reaction Patent Number(s) 4,683,202

  23. Polymerase chain reaction (PCR) a special case of DNA replication in vitro thermocycler

  24. Template DNA unwind DNA polymerase primers dNTPs, Magnesium, proper pH and salt

  25. Template DNA helicase dNTPs Mg++ buffer ( salt, pH) polymerase Template DNA heat (95oC) dNTPs MgCl2 buffer ( salt, pH) Taq polymerase In vitro equivalents

  26. Taq polymerase Heat stable Taq: Thermus aquaticus

  27. Polymerase Chain Reaction

  28. ALU Insertion Chromosome 16 Alu is an example of a so-called "jumping gene" – a transposable DNA sequence that "reproduces" by copying itself and inserting into new chromosome locations. : • First, the inserted Alu is transcribed into messenger RNA by the cellular RNA polymerase. • Then, the mRNA is converted to a double-stranded DNA molecule by reverse transcriptase. • Finally, the DNA copy of Alu is integrated into a new chromosomal locus at the site of a single- or double-stranded break.

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