Genes are composed of nucleic acids (usually DNA)Structures of nucleic acidsA simple view of DNANucleotidesTypes of bases in nucleotidesNucleotides: purine basesBases are attached to C1’ of the sugar via an N-glycosidic bondPhosphate is attached to C5’ of the sugarStructure of a dinucleotideNucleic acids are linear chains of nucleotidesMore on orientation of chains of nucleic acidsBasics of DNA structureDuplex DNAImplications of complementarityBase pairs in DNAMajor types of duplex nucleic acid structuresForms of nucleic acid duplexesHelical parameters for B, A and Z nucleic acidsHyperchromic shift when DNA is denaturedFactors that affect melting temperature, p. 85Electrophoresis to measure SIZEExample of gel electrophoresisGenes are composed of nucleic acids (usually DNA)•Pneumococcus can be transformed from an avirulent to a virulent strain•DNA is the transforming principle•DNA in bacteriophage particles appears in the progeny, but very little protein does.Structures of nucleic acidsNucleotidesDNA structuresSedimentation and ElectrophoresisA simple view of DNAAGCCTCGCATTCGGAGCGTANucleotides•3 components to nucleotides:–Purine or pyrimidine base–Ribose (RNA) or 2-deoxyribose (DNA) sugar –Phosphate•Base + sugar = Nucleoside•Base + sugar + phosphate = NucleotideTypes of bases in nucleotidesPyrimidineNNHONH2CytosineNHNHOOUracilNHNHOOCH3ThymineAmino- Keto-Nucleotides: purine basesNNNNHHNNNNHNH2H2NOAdenineGuanine6-aminopurine A keto-purineBases are attached to C1’ of the sugar via an N-glycosidic bondNNNNNH2OOHCH2HOAdenosine3' 5'1'2’-deoxy-, a nucleosidePhosphate is attached to C5’ of the sugar 1st phosphate is a phosphoester, others are attached as phosphoanhydrides.-O-P-O=ONTP is -O-P-O=O-O-P-OO=OObaseOHOHphosphoanhydridephosphoester-Structure of a dinucleotideThe 3’ C of one nucleotide is linked to the 5’ C of the next nucleotide in a phosphodiester linkage.OOCH2O3' POOOHNNNNNH2OOHCH2OPOOHNNONH25' cytidylyladenylateor5'pCpA5'3'5'Nucleic acids are linear chains of nucleotides•The 3’ C of one nucleotide is linked to the 5’ C of the next nucleotide.•The linkage is by a phosphoester.•The chain has an orientation defined by the sugar-phosphage backbone.•One terminal nucleotide has a “free” 5’ end, and the other has a “free” 3’ end.•Thus we designate orientation by 5’ to 3’.More on orientation of chains of nucleic acids•5’ ACTG 3’ is different from 3’ ACTG 5’•Unless specified otherwise, a chain is written with the 5’ end on the left and the 3’ end on the right.•When complementary strands in DNA are written, usually the top strand is written 5’ to 3’, left to right, and the bottom strand is written 3’ to 5’, left to right.5’ GATTCGTACCG3’ CTAAGCATGGCBasics of DNA structure•2 complementary strands of nucleic acids•Complementarity is based on H-bonding between–Keto bases with amino bases–Pyrimidines with purines•A pairs with T (or U)•G pairs with C•The complementary strands are antiparallel.•The complementary strands are coiled around each other.Duplex DNA•Two strands coil around each other.•Right-handed coils (B form and A.•Coils form major and minor grooves.•Strands have opposite polarity (antiparallel).•Opposing bases in strands are complementary.•Different edges of paired bases are exposed in major and minor grooves.•Sugar-phosphate backbone is on the outside, bases on the inside–B-form DNA: base pairs are close to center of long axis of the duplex.–A-form nucleic acids: base pairs stack away from long axis.Implications of complementarity•One chain (strand) of DNA can serve as the template for synthesis of the complementary chain.•DNA replication: sequence of nucleotides in one chain of the duplex determines the sequence of nucleotides in the other chain.•Transcription: sequence of nucleotides in one chain of the duplex determines the sequence of nucleotides in mRNA or its precursor.Base pairs in DNANNNNOONHHNHHHdeoxy-ribosedeoxy-riboseNNNNNNNNONHHHOCH3deoxy-ribosedeoxy-riboseGuanine : Cytosine Adenine : ThymineMajor grooveMinor grooveMajor grooveMinor grooveMajor types of duplex nucleic acid structures•B form–Most common form of DNA–Right handed DNA-DNA helix–Base pairs stack close to DNA central axis•A form–right handed RNA-DNA and RNA-RNA helix–Base pairs stack away from the central axis •Z form DNA–Repeating purines and pyrimidines–Left-handed helix–May serve as some regulatory signal in cellsForms of nucleic acid duplexesB-form DNAA-form (e.g. duplex RNA)Z DNAHelical parameters for B, A and Z nucleic acidsB A Zhelix sense RH RH LHbp per turn 10 11 12vertical rise per bp 3.4 2.56 3.7 Angstromsrotation per bp +36 +33 -30 degreeshelical diameter 19 23 18 AngstromsHyperchromic shift when DNA is denaturedNative duplex DNADenatured, strands are separate+denaturation by heat orincreasing pHrenaturation by cooling or lowering pHlower A260higher A260hyperchromichypochromic1.01.21.4Tm= melting temperature, midpoint of the transitionTemperatureA260Factors that affect melting temperature, p. 85•The melting tempera-ture (Tm) increases as–Increase G+C–Increase ionic strength (or )•Tm decreases as–Increase denaturants–Increase number of mismatchesTm = 0.41 (% G+C) + 16.6 log M + 81.5 -0.7 (% formamide) -1o (% mismatch)% G+CTmμ = 0.06μ = 0.21Electrophoresis to measure SIZEFor molecules of the same shape, log M is inversely proportional to d.For molecules of the same size, more compact forms, such as supercoiled DNA, moves faster than more extended forms, such as linear DNA.+-Size markersDNA samplesExample of gel electrophoresisAlpha-globingenePCR product217 bpMarkers100200300400 base