Lecture: DNA replication and PCR

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VASUpharm14
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Lecture: DNA replication and PCR
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2010-09-06 12:33:23
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IBHS Skacel part 1
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  1. Who discovered the structure of DNA? not DNA itself
    • Regards to Rosalind Franklin who really deserves the nobel prize. im not bitter.
    • Watson and Crick did in 1953
  2. What did the nature article on DNA provide info on?
    Hydrogen bonding (with A-T, C-G), directionality (antiparallel), replication (mechanism of copying)
  3. DNA stands for
    Deoxyribonucleic acid
  4. RNA stands for
    ribonucleic acid
  5. Nucleic acids are made of nucleotides.
    what 3 things make a nucleotide (monomer)?
    • 1. PO43- (gives the negative charge on nucleic acids)
    • 2. pentose sugar
    • 3. nitrogenous base
  6. DNA vs RNA
    in Pentose sugar
    • 2' is either H or OH
  7. Name the bonds formed between monomers of Nucleic acids.
    Phosphodiester bonds (Bond is between 5' and 3')
  8. What are the cyclic nitrogen-containing bases?
    in RNA and DNA and hydrogen bonding number?
    • RNA: A,C,G,U
    • DNA: A,C,G,T
    • A-T = 2 H+ bond
    • G-C = 3 H+ bond
  9. DNA directionality
    parallel or antiparallel?
    • antiparallel
    • (imagine knuckle on hand, each bump is a sugar connecting to fingers (base), the in between knuckle is phosphate group. use both hands and grap onto fingers. one hand should be upside down. the thumbs point in the directions. the directions are opposite. thus antiparallel).
  10. T/F
    only the upper strand can serve as template
    • False!
    • both can serve as template
  11. Replication is semi-conservative.
    what does this mean?
    • DNA replication = 2(1 parent with 1 daughter)
    • like the parent trap movie. the two lindsey lohans split with their mom or dad. (but really, there's only one lindsey lohan in that movie)
  12. DNA template is read in what direction?
    DNA is synthesized in what direction?
    • 1. read 3' to 5'
    • 2. synthesized 5' to 3'
  13. DNA synthesis begins where?
    replication bubbles (origins)
  14. How many forks in a replication bubble in DNA replication?
    2, in opposite directions
  15. Replication requirements in DNA (5)
    • 1. replication bubble
    • 2. DNA polymerase (adds to 3' end)
    • 3. template
    • 4. PRIMER
    • 5. 4 dNTPs
  16. where does energy come from to do replication?
    cleavage between alpha and beta phosphates on previous dNTP
  17. Leading strand
    • synthesized continuously
    • toward the fork
  18. lagging strand
    • synthesized discontinuously (Okazaki fragments)
    • away from fork
  19. Helicase
    • breaks HYDROGEN bonds to separate strands of DNA.
    • unwinds
    • rotary engine - goes fast (1000nt per second), use ATP
    • 6 subunits
  20. Single Strand binding proteins
    AKA helix destabilizing proteins
    AKA replication protein A (RPA) - in Eukaryotes!
    • keep strands separate (no hairpins or helices formed)
    • doesn't cover the bases
  21. context: torsional stress
    Topoisomerase 1
    • for 1 tangled piece of double stranded DNA
    • reversible nuclease that nicks a phosphodiester bond
  22. context: torsional stress
    Topoisomerase 2
    • for 2 tangled pieces of double stranded DNA
    • reversible nuclease that breaks a helix.
  23. DNA polymerase needs 2 things to do replication
    • 1. primer
    • 2. free 3'OH
  24. context: RNA primers
    what makes RNA primers
    • DNA primase use DNA template with rNTPs.
    • free 3' OH on RNA primer to synthesize DNA.
    • MANY primers in lagging strand vs ONE in leading strand.
  25. context: lagging strand
    Ribonuclease H
    removes RNA primer then adds DNA
  26. context: lagging strand
    DNA ligase
    • seals the Okazaki fragments together
    • -creates phosphodiester bonds
    • -needs ATP
    • smaller fragments in Eukaryotes than Prokaryotes
  27. context: lagging strand
    DNA polymerase
    • alpha
    • -bigger than delta (leading)
  28. context: leading strand
    DNA polymerase
    • delta
    • -smaller than alpha (lagging)
  29. context: accessory proteins
    PCNA
    • -proliferating cell nuclear antigen
    • sliding clamp/ polymerase clamp
    • keeps DNA polymerase on longer. (processive)
  30. context: accessory proteins
    RFC
    • replication factor c
    • clamp LOADER for PCNA
  31. T/F
    leading and lagging strands are COUPLED
    • true dat :D
    • never leave your friends
  32. Telomeres
    • ends of DNA chromosomes
    • required for replication and stability
    • 3' repeated sequence overhang.
    • ex: shoelaces protected ends.
  33. Telomerase
    • = enzyme (reverse transcriptase) + RNA complex (ribonucleoprotein)
    • the lagging strand doesn't have enough room for final Okazaki fragment
    • Telomerase adds extra template using own RNA template
    • polymerase alpha finishes job
  34. context: telomeres
    Nuclease
    • chews back 5' end of chromosome
    • protruding 3' end makes T-LOOP
  35. T/F
    somatic cells have LOW telomerase activity
    • TRUE :(
    • except bone marrow (hematopoietic) and skin cells (epithelia) (yay! :D)
  36. context: Prokaryotic replication
    kind of chromosome
    double stranded and circular
  37. context: Prokaryotic replication
    how many bubbles/origins of replication
    1 = OriC
  38. context: Prokaryotic replication
    how to find the Ori C
    • initiator proteins bind to A-T rich site
    • attracts DNA helicase + helix loader (like clamp loader!)
  39. context: Prokaryotic replication
    what kind of polymerase?
    DNA polymerase 3
  40. context: Prokaryotic replication
    Replication Refractory period
    • no replication unless A is methylated
    • with DAM METHYLASE
    • (damn methalyase, i need you to replicate).
  41. context: Prokaryotic replication
    Termination
    • use termination sequence (TER AND TUS)
    • TUS PROTEINS bind to TER SITES to stop helicase
    • TOPO 2 release the covalently bond strands
  42. DNA proofreading
    • DNA polymerase has this capability
    • error rates very small
  43. context: tautomers
    keto
    • H+ binds to Nitrogen in ring
    • normal binding
  44. context: tautomers
    enol
    • H+ binds to Oxygen outside of ring
    • not normal: will allow weird binding ex: G=T
    • DNA polymerase should exonuclease this
  45. context: other repair mechanisms when proofreading fails
    Strand directed MISMATCH pair
    • nicks on daughter strand (MUT L) and kinks on pair strands (MUT S) detected
    • then removed piece
    • MUT H in prokaryotes know that daughter strand is not methylated
  46. PCR history
    • polymerase chain reaction
    • invitro to copy/amplify DNA
    • discovered by Kary Mullis
    • for cloning and crime investigation
  47. PCR: what is needed in the tube (6)
    • 1. template
    • 2. primers
    • 3. DNA polymerase (Taq- for high temperatures)
    • 4. dNTPs
    • 5. Buffer
    • 6. Magnesium ions
  48. 3 major steps in PCR
    • 1. denaturation (95oC)
    • 2. annealing (55oC) the oligos primers
    • 3. elongation (72oC)
    • T4 DNA ligase!
  49. results of PCR
    exponential amounts!
  50. Restriction enzymes
    • cut double stranded DNA
    • palindromes
    • sticky or blunt end cuts
    • found in bacteria - for cutting out viral DNA (not methylated)
  51. plasmids
    • extrachromosomal circular DNA
    • 3 important features:
    • 1. origin of replication
    • 2. MCS (restriction enzymes)
    • 3. antibiotic resistance genes
  52. Recombinant DNA
    • 2 DNA sources linked
    • use restriction enzymes
    • then ligase
    • -can use bacteria to amplify by putting in plasmid vector

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