Dna replication and pcr

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  • Dna replication and pcr

    1. 1. Genetics: Analysis and Principles By Robert J. Brooker CHAPTER 11 DNA replication
    2. 2. DNA Structure Helps Explain How It Duplicates <ul><li>DNA is two nucleotide strands held together by hydrogen bonds </li></ul><ul><li>Hydrogen bonds between two strands are easily broken </li></ul><ul><li>Each single strand then serves as template for new strand </li></ul>
    3. 3. DNA Replication <ul><li>Each old strand stays intact </li></ul><ul><li>Each new DNA molecule is half “old” and half “new” </li></ul>Fig. 1-7, p.212
    4. 4. DNA is a double helix A sugar and phosphate “ backbone” connects nucleotides in a chain. Two nucleotide chains together wind into a helix. DNA strands are antiparallel. DNA has directionality. Hydrogen bonds between paired bases hold the two DNA strands together. P A P C P G P T P C P G P A P C P T G P P C P P G P 5’ 3’ 3’ 5’
    5. 5. DNA Melting Temperature
    6. 6. Orientation of DNA <ul><li>The directionality of a DNA strand is due to the orientation of the phosphate-sugar backbone. </li></ul>The carbon atoms on the sugar ring are numbered for reference. The 5’ and 3’ hydroxyl groups (highlighted on the left) are used to attach phosphate groups.
    7. 7. <ul><li>In the late 1950s, three different mechanisms were proposed for the replication of DNA </li></ul><ul><ul><li>Conservative model </li></ul></ul><ul><ul><ul><li>Both parental strands stay together after DNA replication </li></ul></ul></ul><ul><ul><li>Semiconservative model </li></ul></ul><ul><ul><ul><li>The double-stranded DNA contains one parental and one daughter strand following replication </li></ul></ul></ul><ul><ul><li>Dispersive model </li></ul></ul><ul><ul><ul><li>Parental and daughter DNA are interspersed in both strands following replication </li></ul></ul></ul>Experiment : Which Model of DNA Replication is Correct? Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 11-5
    8. 8. Figure 11.2 11-6
    9. 9. <ul><li>In 1958, Matthew Meselson and Franklin Stahl devised a method to investigate these models </li></ul><ul><ul><li>They found a way to experimentally distinguish between daughter and parental strands </li></ul></ul><ul><li>Their experiment can be summarized as such </li></ul><ul><ul><li>Grow E. coli in the presence of 15 N (a heavy isotope of Nitrogen) for many generations </li></ul></ul><ul><ul><ul><li>The population of cells had heavy-labeled DNA </li></ul></ul></ul><ul><ul><li>Switch E. coli to medium containing only 14 N (a light isotope of Nitrogen) </li></ul></ul><ul><ul><li>Collect sample of cells after various times </li></ul></ul><ul><ul><li>Analyze the density of the DNA by centrifugation using a CsCl gradient </li></ul></ul>Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 11-7
    10. 10. The Hypothesis <ul><ul><li>This experiment aims to determine which of the three models of DNA replication is correct </li></ul></ul>Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Testing the Hypothesis T <ul><ul><li>Refer to Figure 11.3 </li></ul></ul>11-8
    11. 11. 11-9 Figure 11.3
    12. 12. Figure 11.3 11-10
    13. 13. The Data Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 11-11
    14. 14. Interpreting the Data Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 11-12 After one generation, DNA is “half-heavy” This is consistent with both semi-conservative and dispersive models After ~ two generations, DNA is of two types: “light” and “half-heavy” This is consistent with only the semi-conservative model
    15. 15. Meselson-Stahl Experiment Conclusion: Replication is semiconservative.
    16. 16. Important rules about DNA/RNA Thus:: AGCTCCGCTA means 5’ AGCTCCGCTA 3’ The complementary strand of 5’ AGCTCCGCTA 3’ Is then 5’ TAGCGGAGCT 3’ !!!!!!!!! 1. The base sequence is always shown in 5’ to 3’ direction
    17. 17. Replicatoin can’t just start: 1. All DNA polymerases need a primer 2. The primer can be a piece of RNA or DNA 3. It must be “base-paired” with the “template” and with 3’OH Thus: 3’ 5’ 5’ 3’ Template strand primer Synthesis direction
    18. 18. A Closer Look at Strand Assembly <ul><li>Energy for strand assembly is provided by removal of two phosphate groups from free nucleotides </li></ul>newly forming DNA strand one parent DNA strand
    19. 19. “ Proof-reading” is essential at DNA replication <ul><li>What is proof-reading? </li></ul>1. If there is a wrong base built in, then there is no base paring possible. 2. The DNA polymerase can’t continue on building in the next base. 3. The DNA polymerase removes the “wrong” base and starts over
    20. 20. Klenow Fragment (of pol I) (The proof-reading activity)
    21. 21. DNA Polymerases <ul><li>DNA pol I </li></ul><ul><ul><li>Composed of a single polypeptide </li></ul></ul><ul><ul><li>Removes the RNA primers and replaces them with DNA </li></ul></ul><ul><li>DNA pol III </li></ul><ul><ul><li>Responsible for most of the DNA replication </li></ul></ul><ul><ul><li>Composed of 10 different subunits (Table 11.2) </li></ul></ul><ul><ul><ul><li>The  subunit synthesizes DNA </li></ul></ul></ul><ul><ul><ul><li>The other 9 fulfill other functions </li></ul></ul></ul><ul><ul><li>The complex of all 10 is referred to as the DNA pol III holoenzyme </li></ul></ul>There are 3 others pol II, IV and V, involved in repair (no details)
    22. 22. <ul><li>Eukaryotes have ~ 15 DNA polymerases ! </li></ul><ul><li>Each polymerase has its own function! </li></ul><ul><ul><li>DNA synthesis during genome replication (cell division) </li></ul></ul><ul><ul><li>Other are involved in DNA damage repair </li></ul></ul>
    23. 23. <ul><li>Bacterial DNA polymerases may vary in their subunit composition </li></ul><ul><ul><li>However, they have the same type of catalytic subunit </li></ul></ul>Structure resembles a human right hand Template DNA thread through the palm; Thumb and fingers wrapped around the DNA
    24. 24. <ul><li>The figure presents an overview of the process of bacterial chromosome replication </li></ul><ul><ul><li>DNA synthesis begins at a site termed the origin of replication </li></ul></ul><ul><ul><ul><li>Each bacterial chromosome has only one </li></ul></ul></ul><ul><ul><li>Synthesis of DNA proceeds bidirectionally around the bacterial chromosome </li></ul></ul><ul><ul><li>The replication forks eventually meet at the opposite side of the bacterial chromosome </li></ul></ul><ul><ul><ul><li>This ends replication </li></ul></ul></ul>BACTERIAL DNA REPLICATION
    25. 25. Initiation of Replication <ul><li>The origin of replication in E. coli is termed oriC </li></ul><ul><ul><li>ori gin of C hromosomal replication </li></ul></ul><ul><li>Three types of DNA sequences in oriC are functionally significant </li></ul><ul><ul><li>AT-rich region </li></ul></ul><ul><ul><li>DnaA boxes </li></ul></ul><ul><ul><li>GATC methylation sites </li></ul></ul>
    26. 27. Proteins involved in E. coli replication Terms to be known
    27. 28. Why the discontinuous additions? Nucleotides can only be joined to an exposed —OH group that is attached to the 3’ carbon of a growing strand. Strand Assembly
    28. 29. Continuous and Discontinuous Assembly
    29. 30. Replication Helicase protein binds to DNA sequences called origins and unwinds DNA strands. Binding proteins prevent single strands from rewinding. 5’ 3’ 5’ 3’ Primase protein makes a short segment of RNA complementary to the DNA, a primer. 3’ 5’ 5’ 3’
    30. 31. Replication DNA polymerase enzyme adds DNA nucleotides to the RNA primer. Overall direction of replication 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’
    31. 32. Replication DNA polymerase enzyme adds DNA nucleotides to the RNA primer. DNA polymerase proofreads bases added and replaces incorrect nucleotides. 5’ 5’ Overall direction of replication 5’ 3’ 5’ 3’ 3’ 3’
    32. 33. Replication Leading strand synthesis continues in a 5’ to 3’ direction. 5’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ Overall direction of replication
    33. 34. Replication Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments. 3’ 5’ 5’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ Overall direction of replication Okazaki fragment
    34. 35. Replication 5’ 5’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ Overall direction of replication 3’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments. Okazaki fragment
    35. 36. Replication 5’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ 3’ 5’ 5’ 3’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
    36. 37. Replication 3’ 5’ 3’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments. 5’ 5’ 3’ 5’ 3’ 3’ 5’ 5’ 3’
    37. 38. Replication Exonuclease enzymes remove RNA primers. 5’ 5’ 3’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’
    38. 39. Replication Exonuclease enzymes remove RNA primers. Ligase forms bonds between sugar-phosphate backbone. 3’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’
    39. 40. Enzymes in Replication <ul><li>Enzymes ( Helicases ) unwind the two strands </li></ul><ul><li>DNA polymerase needed for the synthesis of complementary strand </li></ul><ul><li>DNA ligase joins pieces of the lagging strand together </li></ul>
    40. 41. Enzymes in DNA replication Helicase unwinds parental double helix Binding proteins stabilize separate strands DNA polymerase binds nucleotides to form new strands Ligase joins Okazaki fragments and seals other nicks in sugar-phosphate backbone Primase adds short primer to template strand Exonuclease removes RNA primer and inserts the correct bases
    41. 42. DNA replication mistakes <ul><li>The most errors in DNA sequence occur during replication. </li></ul><ul><li>Reparation takes place after replication is finished </li></ul><ul><li>DNA polymerases can get the right sequence from the complementary strand and repair, along with DNA ligase, the wrong bases. </li></ul>
    42. 43. <ul><li>Eukaryotic DNA replication is not as well understood as bacterial replication </li></ul><ul><ul><li>The two processes do have extensive similarities, </li></ul></ul><ul><ul><ul><li>The bacterial enzymes described before have also been found in eukaryotes </li></ul></ul></ul><ul><ul><li>Nevertheless, DNA replication in eukaryotes is more complex </li></ul></ul><ul><ul><ul><li>Large linear chromosomes </li></ul></ul></ul><ul><ul><ul><li>Tight packaging within nucleosomes </li></ul></ul></ul><ul><ul><ul><li>More complicated cell cycle regulation </li></ul></ul></ul>EUKARYOTIC DNA REPLICATION
    43. 44. Multiple origins of replication in eukaryotes <ul><ul><li>Origins of replication in Saccharomyces cerevisiae </li></ul></ul><ul><ul><li>are termed ARS </li></ul></ul><ul><ul><li>elements ( A utonomously R eplicating S equence) </li></ul></ul><ul><ul><ul><li>They are 100-150 bp in length </li></ul></ul></ul><ul><ul><ul><li>They have a high percentage of A and T </li></ul></ul></ul><ul><ul><ul><li>They have three or four copies of a specific </li></ul></ul></ul><ul><ul><ul><li>sequence </li></ul></ul></ul><ul><ul><ul><li>The replication rate is similar to E. coli </li></ul></ul></ul>
    44. 45. Telomeres and DNA Replication <ul><li>Linear eukaryotic chromosomes have telomeres at both ends </li></ul><ul><li>The term telomere refers to the complex of telomeric DNA sequences and bound proteins </li></ul>
    45. 46. <ul><li>Telomeric sequences consist of </li></ul><ul><ul><li>Moderately repetitive tandem arrays </li></ul></ul><ul><ul><li>3’ overhang that is 12-16 nucleotides long </li></ul></ul><ul><li>Telomeric sequence typically for ‘vertebrates’: TTAGGG </li></ul><ul><li>See: http://telomerase.asu.edu/sequences.html </li></ul>
    46. 47. <ul><li>DNA polymerases possess two unusual features </li></ul><ul><ul><li>1. They synthesize DNA only in the 5’ to 3’ direction </li></ul></ul><ul><ul><li>2. They cannot initiate DNA synthesis </li></ul></ul><ul><li>These two features pose a problem at the 3’ end of linear chromosomes-the end of the strand cannot be replicated! </li></ul>
    47. 48. <ul><li>That is why the ‘normal’ differentiated cells have linear chromosomes that become shorter and shorter and thus these cells have an ending life. </li></ul><ul><ul><li>Only cancer and stam cells can solve this problem by expression of a telomerase enzyme. </li></ul></ul><ul><li>The telomerase contains protein and RNA </li></ul><ul><ul><li>The RNA is complementary to the DNA sequence of the telomere ‘repeat’ sequence. </li></ul></ul><ul><ul><ul><li>Therefore, it can bind to the 3’-end of the chromosomes. </li></ul></ul></ul><ul><ul><li>Next slide will give more explination </li></ul></ul>
    48. 49. Step 1 = Binding Step 3 = Translocation The binding-polymerization-translocation cycle can occurs many times This greatly lengthens one of the strands RNA primer Step 2 = Polymerization The end is now lengthened
    49. 50. Polymerase Chain Reaction (PCR) Is a method in which multiple repetitions of DNA replication are performed in a test tube. Mix in test tube: DNA template DNA to be amplified Primers one complementary to each strand Nucleotides dATP,d GTP, dCTP, and dTTP DNA polymerase heat stable form from thermophilic bacteria
    50. 51. Polymerase Chain Reaction (PCR) DNA template is denatured with heat to separate strands. C T T G A T C G C 3’ 5’ G A T C A A G C G 3’ 5’
    51. 52. Polymerase Chain Reaction (PCR) DNA template is denatured with heat to separate strands. C T T G A T C G C 3’ 5’ G A T C A A G C G 3’ 5’
    52. 53. Polymerase Chain Reaction (PCR) C T T G A T C G C 3’ 5’ G A T C A A G C G 3’ 5’ Each DNA primer anneals, binding to its complementary sequence on the template DNA C T T G C G 5’ 5’ 3’ 3’
    53. 54. Polymerase Chain Reaction (PCR) DNA polymerase creates a new strand of DNA complementary to the template DNA starting from the primer. C T T G A T C G C 3’ 5’ G A T C A A G C G 3’ 5’ C T T G C G 5’ 5’ 3’ C G C G A T G A A C T A 3’
    54. 55. Polymerase Chain Reaction (PCR) Denaturation Each DNA primer anneals, binding to its complementary sequence on the template DNA DNA template is denatured with high heat to separate strands. Annealing Extension DNA polymerase creates a new strand of DNA complementary to the template DNA starting from the primer. Multiple rounds of denaturation-annealing-extension are performed to create many copies of the template DNA between the two primer sequences.
    55. 56. Polymerase Chain Reaction (PCR) After 35 cycles a single molecule will be amplified 2 35 times = ± 50.000.000.000 x
    56. 57. Visualization of PCR products
    57. 58. agarose gel-electrophoresis of DNA Top Bottom Slab of agarose gel ( ethidium bromide staining ) Positive pole Negative pole DNA is Negatively charged Small molecules dissolve faster separation based on size
    58. 59. <ul><li>Structural Overview of DNA Replication </li></ul><ul><ul><li>Existing DNA strands act as templates for the synthesis of new strands </li></ul></ul><ul><ul><li>Three different models were proposed that described the net result of DNA replication </li></ul></ul><ul><li>Bacterial DNA Replication </li></ul><ul><ul><li>Bacterial chromosomes contain a single origin of replication </li></ul></ul><ul><ul><li>Replication is initiated by the binding of DnaA protein to the origin of replication </li></ul></ul><ul><ul><li>Several proteins are required for DNA replication at the replication fork </li></ul></ul><ul><ul><li>DNA polymerase III is a processive enzyme that uses deoxyribonucleoside triphosphates </li></ul></ul><ul><ul><li>Replication is terminated when the replication forks meet at the terminus sequences </li></ul></ul><ul><ul><li>Certain enzymes of DNA replication bind to each other to form a complex </li></ul></ul><ul><ul><li>The fidelity of DNA replication is ensured by proofreading mechanisms </li></ul></ul><ul><ul><li>Bacterial DNA replication is coordinated with cell division </li></ul></ul><ul><ul><li>DNA replication can be studied in vitro </li></ul></ul><ul><ul><li>The isolation of mutants has been instrumental in our understanding of DNA replication </li></ul></ul><ul><li>Eukaryotic DNA replication </li></ul><ul><ul><li>Initiation occurs at multiple origins of replication on linear eukaryotic chromosomes </li></ul></ul><ul><ul><li>Eukaryotes contain several different DNA polymerases </li></ul></ul><ul><ul><li>The ends of eukaryotic chromosomes are replicated by telomerase </li></ul></ul>Study outline:

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