Structures of nucleic acids IISouthern blot-hybridizationsSteps in Southern blot-hybridizationSteps in Southern blot-hybridization, continuedStrategy to determine DNA or RNA sequenceCommon sequencing techniquesExample of dideoxynucleotide sequencingExample of output from automated dideoxysequencingSupercoiling of topologically constrained DNADifferent topological forms of DNANegative and positive supercoilsComponents of DNA Topology : TwistComponents of DNA Topology : WritheComponents of DNA Topology : Linking numberL cannot change unless one or both strands are broken and reformedRelationship between supercoiling and twistingDNA in most cells is negatively supercoiledNegatively supercoiled DNA favors unwindingTopoisomerase ITopoisomerase I: nicking & closingTopoisomerase IITopoII: double strand break and passageStructures of nucleic acids IISouthern blot-hybridizationsSequencingSupercoiling: Twisting, Writhing and Linking numberSouthern 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.Steps in Southern blot-hybridizationSeparate restriction fragmentsby size using electrophoresisthrough an agarose gel.Denature duplex DNATransfer denatured DNA fragments ontoa membrane to produce an immobilized replica of the gel-separated fragments (the "blot").nylon or nitrocellulosemembrane*********+Incubate the blot (with denatured DNAfragments immobilized) with an excess of labeled DNA from a specific gene or region under conditions that favor formation of specific hybrids.Wash off any non-specifically bound probe.The probe has now hybridized to therestriction fragments that have the gene of interest. ****** This pattern of hybridization to the blot can be detected by exposure to X-ray film or phosphor screens.Image on the exposed film after developing. This tells you which restriction fragments have the gene or region of interest (if you remembered to include size markers!).Steps in Southern blot-hybridization, continuedStrategy 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 TSequence is …GAT….Common sequencing techniquesDNA: Maxam& GilbertDNA: SangerRNAChain terminationby dideoxy-nucleotidesBase-specific chemical cleavageNucleotide-specific enzymatic cleavageRestriction endonucleasePrimer for DNA polymeraseNatural end of RNA32P32P or fluores-cence32PTechnique Common end Label Nt-specific endExample of dideoxynucleotide sequencing•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 output from automated dideoxysequencingSupercoiling of topologically constrained DNA•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.Different topological forms of DNAGenes VI : Figure 5-9Negative and positive supercoils•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.Components of DNA Topology : Twist•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 : Writhe•W = Writhing Number •Refers to the turning of the axis of the DNA duplex in space•Number of times the duplex DNA crosses over itselfRelaxed molecule W=0Negative supercoils, W is negativePositive supercoils, W is positiveComponents of DNA Topology : Linking number•L = Linking Number = total number of times one strand of the double helix (of a closed molecule) encircles (or links) the other.•L = W + TL cannot change unless one or both strands are broken and reformed•A change in the linking number, L, is partitioned between T and W, i.e.•L=W+T• if L = 0, then W= -TRelationship between supercoiling and twistingFigure from M. Gellert; Kornberg and BakerDNA in most cells is negatively supercoiled•The superhelical density is simply the number of superhelical (S.H.) turns per turn (or twist) of double helix.•Superhelical density =  = 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)Negatively supercoiled DNA favors unwinding•Negative supercoiled DNA has energy stored that favors unwinding, or a transition from B-form to Z DNA.•For  = -0.05, G=-9 Kcal/mole favoring unwindingThus negative supercoiling could favor initiation of transcription and initiation of replication.Topoisomerase I•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: nicking & closingGenes VI : Figure 17-15One strand passes through a nick in the other strand.Topoisomerase II•Topo II = gyrase•Uses the energy of ATP hydrolysis to introduce negative supercoils•Its mechanism of action is to make a transient double