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Structures of nucleic acids II

Structures of nucleic acids II. Southern blot-hybridizations Sequencing Supercoiling: Twisting, Writhing and Linking number. Southern blot-hybridizations. Allows the detection of a particular DNA sequence among the many displayed on an electrophoretic gel.

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Structures of nucleic acids II

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  1. Structures of nucleic acids II Southern blot-hybridizations Sequencing Supercoiling: Twisting, Writhing and Linking number

  2. Southern blot-hybridizations • Allows the detection of a particular DNA sequence among the many displayed on an electrophoretic gel. • e.g. determine which among many restriction fragments contains a gene. • Transfer the size-separated DNA fragments out of the agarose gel and onto a membrane (nylon or nitrocellulose) to make an immobilized replica of the gel pattern. • Hybridize the membrane to a specific, labeled nucleic acid probe and determine which DNA fragments contain that labeled sequence.

  3. Steps in Southern blot-hybridization

  4. Steps in Southern blot-hybridization, continued

  5. Strategy to determine DNA or RNA sequence • Generate a nested set of fragments with one common, labeled end • The other end terminates at one of the 4 nucleotides • Electrophoretic resolution of the fragments allows the reading of the sequence: Fragment of length 47 ends at G 48 A 49 T Sequence is …GAT….

  6. Common sequencing techniques Technique Common end Label Nt-specific end DNA: Maxam & Gilbert Restriction endonuclease 32P Base-specific chemical cleavage DNA: Sanger Primer for DNA polymerase 32P or fluores-cence Chain termination by dideoxy-nucleotides RNA Natural end of RNA 32P Nucleotide-specific enzymatic cleavage

  7. Reactions: Fig. 2.30 Output: Fig. 2.31 Cycle Sequencing Movie: http://vector.cshl.org/resources/BiologyAnimationLibrary.htm The Sanger dideoxynucleotide method is amenable to automation performed by robots. This approach is the one adapted for virtually all the whole-genome sequencing projects. Example of dideoxynucleotide sequencing

  8. Example of output from automated dideoxysequencing

  9. Topologically closed DNA can be circular (covalently closed circles) or loops that are constrained at the base The coiling (or wrapping) of duplex DNA around its own axis is called supercoiling. Supercoiling of topologically constrained DNA

  10. Different topological forms of DNA Genes VI : Figure 5-9

  11. Negative supercoils twist the DNA about its axis in the opposite direction from the clockwise turns of the right-handed (R-H) double helix. Underwound (favors unwinding of duplex). Has right-handed supercoil turns. Positive supercoils twist the DNA in the same direction as the turns of the R-H double helix. Overwound (helix is wound more tightly). Has left-handed supercoil turns. Negative and positive supercoils

  12. The clockwise turns of R-H double helix generate a positive Twist (T). The counterclockwise turns of L-H helix (Z form) generate a negative T. T = Twisting Number B form DNA: + (# bp/10 bp per twist) A form NA: + (# bp/11 bp per twist) Z DNA: - (# bp/12 bp per twist) Components of DNA Topology : Twist

  13. W = Writhing Number Refers to the turning of the axis of the DNA duplex in space Number of times the duplex DNA crosses over itself Relaxed molecule W=0 Negative supercoils, W is negative Positive supercoils, W is positive Components of DNA Topology : Writhe

  14. L = Linking Number = total number of times one strand of the double helix (of a closed molecule) encircles (or links) the other. L = W + T Components of DNA Topology : Linking number

  15. A change in the linking number, DL, is partitioned between T and W, i.e. DL=DW+DT if DL = 0, thenDW= -DT L cannot change unless one or both strands are broken and reformed

  16. Relationship between supercoiling and twisting Figure from M. Gellert; Kornberg and Baker

  17. The superhelical density is simply the number of superhelical (S.H.) turns per turn (or twist) of double helix. Superhelical density = s = W/T = -0.05 for natural bacterial DNA i.e., in bacterial DNA, there is 1 negative S.H. turn per 200 bp (calculated from 1 negative S.H. turn per 20 twists = 1 negative S.H. turn per 200 bp) DNA in most cells is negatively supercoiled

  18. Negative supercoiled DNA has energy stored that favors unwinding, or a transition from B-form to Z DNA. For s = -0.05, DG=-9 Kcal/mole favoring unwindingThus negative supercoiling could favor initiation of transcription and initiation of replication. Negatively supercoiled DNA favors unwinding

  19. Topoisomerases: catalyze a change in the Linking Number of DNA Topo I = nicking-closing enzyme, can relax positive or negative supercoiled DNA Makes a transient break in 1 strand E. coli Topo I specifically relaxes negatively supercoiled DNA. Calf thymus Topo I works on both negatively and positively supercoiled DNA. Topoisomerase I

  20. Topoisomerase I: nicking & closing One strand passes through a nick in the other strand. Genes VI : Figure 17-15

  21. Topo II = gyrase Uses the energy of ATP hydrolysis to introduce negative supercoils Its mechanism of action is to make a transient double strand break, pass a duplex DNA through the break, and then re-seal the break. Topoisomerase II

  22. TopoII: double strand break and passage

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