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Original report by Watson and Crick describing theirprediction ofDNA structureNature, April 25, 1953Nucleotide building blocksbasesugarRNADNAmono, di, or triphosphateCanonical bases in DNA and RNARNAonlyDNAonlychemically reactivephosphodiester linkage &glycosidic bondImportant features of a polynucleotide chainbackbone hasnegative chargebases have hydrogen bondacceptors and donorsthe chain has polarity: 5’-3’Watson-Crick model for DNA structure3. Duplex DNA strandsare antiparallel andcomplementary.Backbone outside;H-bonded basesstacked inside.2. Strand has5’-3’ polarity1. Nucleotide unit4. The strands twist in a right-handed helixCommon geometry for all 4 base pairsC G T ASimilar widthSimilar angle of base from sugar (glycosidic bond)G:C has 3 H-bonds while A:T has 2 H-bonds• A-T and G-C base pairs• 2 strands with anti-parallel polarity• bases in, backbone out• right-handed helical twistALSO:• bases perpendicular to helix axis• major and minor grooves• 10 bp/36 angstrom helical repeat• 20 angstrom wide: bp cross centerB-form dsDNA features:major grooveminor grooveBase pairing: ‘horizontal’ in this viewBase stacking: ‘vertical’ in this viewElectrostatic repulsion of phosphate backbone:gives a helical twistEnergetic motivationsfor structure formation:Denature/melt/unpairand Renature/anneal/hybridize• Double-stranded DNA is thermodynamically morestable than the separated strands (underphysiological conditions)• Complete unpairing of the 2 strands is denaturation;the reverse reaction is renaturation• Heat or chaotropic agents (urea) promotedenaturation; removing them promotes renaturation• Melting point (Tm) - temperature at which 1/2 ofthe DNA has become single stranded• Melting curves can be followed at Abs260nmMelting curveDenatured DNA absorbs more than dsDNARNA forms base-pairs: secondary structureSome of the modified bases found in RNA,produced by post-transcriptional reactions.A tRNA molecule(A) The cloverleaf secondary structure(B, C) The L-shaped tertiary structure,based on x-ray diffraction analysis.(D) Primary sequenceThree-dimensional structureof phenylalanine tRNAAveraged structure of A form helixRNA adopts this form due to ribose 2’-OHBall-and-stick Space filling 3’Minor groove (shallow and wide) Major groove (deep and narrow) Twist/bp ~32.7°~11 bp/turn5’Bases tilted~20°0.26 nM rise/bpAveraged structure of B form helixBall-and-stick Space filling Minor groove (narrow) Major groove (wide) 5’ 3’Twist/bp ~34°~10 bp/turnBases tilted~0°0.34 nM rise/bpDNA prefers B form but can adopt A formMinor grooveMajor grooveComparison of helical parameters• B-form major groove depth is accessible to protein side chains, while A-form major groove is too deep: proteins can bind dsRNAsequence-specifically without distorting the helix.• A-form is more thermodynamically stable per base-pair:shorter duplex needed for stable RNA secondary structure.Restriction enzymes cut dsDNAat specific sequences: restriction sitesEcoRI reads dsDNA in the major groove• Homodimer of 2 identicalprotein subunits (purple andyellow)• Bound to a palindromic DNAsequence (same sequence onblue and green strands)looking into the minor groovelooking into the major grooverotation of 180 degrees (back side):Many restriction enzymes have been identified• Three types of ends: 5’ overhang, blunt and 3’ overhang• Cognate methyl transferases protect host genome from digestion;Restriction-modification systems degrade “foreign” DNA.Gel electrophoresis separates DNA by sizeAgarose: big fragments (>300 bp)Acrylamide: smaller fragmentsMobility proportional to log MW.Common DNA fluorescent dyesSouthern blot to detect sequence similarityDNA restriction fragments or other forms of DNA areresolved by agarose gel electrophoresis.The fragments are denatured to single strands. The gel isplaced in buffer and covered by a nitrocellulose filterand a stack of paper towels. DNA is carried to thefilter by the buffer, which is wicked up by the towels.The DNA-bound filter is incubated with a denatured,labeled DNA probe. Strands are allowed to reanneal.If probe is complementary to the sample DNA, thefilter will remain labeled when unbound probe is


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