Slide 1Lecture OutlineSome Methods for Studying DNASpectrophotometry of DNASlide 5Denaturation of DNADNA Melting CurvesSlide 8DNA Renaturation or Annealing Denaturation is reversible!Sequences do not have to be identical to renatureSlide 11NucleasesRestriction Endonucleases “Restriction Enzymes”Restriction Endonucleases (= “Restriction Enzyme”)Restriction EnzymesSlide 16Slide 17Why “Restriction” Endonuclease?Slide 19Uses of Restriction EnzymesDNA Separation by Gel ElectrophoresisSeparation of DNA Fragments by Gel ElectrophoresisSlide 23The Gel Sieves the Molecules - Smaller Molecules Migrate FasterGel ElectrophoresisGel ElectrophoresisSlide 27Slide 28Slide 29Slide 30Slide 31Restriction AnalysisSlide 33Information from Gel ElectrophoresisSlide 35Slide 36Southern BlotsSlide 38Slide 39Southern BlotMCB 250 - Lecture 5Methods for studying DNALecture Outline•DNA Denaturation•DNA denaturation and renaturation - hybridization•Restriction endonucleases•Separation of DNA fragments by electrophoresis•Identification of specific DNA molecules by hybridization - Southern blotsSome Methods for Studying DNASpectrophotometry of DNAThe Hyperchromic Effect Wavelength (nm)AbsorbanceNativeDenatured200 250 260 3001.51.00.50Fig 4-15Denaturation of DNA•The DNA strands are held together by weak non-covalent interactions•The strands will separate or “denature” if these interactions are disrupted –High temperature causes strands to separate (denature). Tm: melting temperature. Tm depends on base composition:high GC has a higher Tm.–Hydrogen bonding reagents – Urea–pH>11 –Organic solvents, e.g., methanol–Decreased salt concentration•Base stacking is cooperative–Ends of the DNA “breathe” (not annealed)–ssDNA binding proteins can denatureDNA Melting CurvesAbsorbance1.01.21.4Figure 6-293 μmDNA Renaturation or AnnealingDenaturation is reversible!Sequences do not have to be identical to renature•Duplex DNA in which one strand differs from the other are called heteroduplexes (as opposed to homoduplexes).•The heteroduplexes are hybrid molecules and the process of forming them is called hybridization.Hybrid moleculesFig 4-14Nucleases•Nucleases are enzymes that cut nucleic acids by hydrolyzing phosphodiester bonds•DNase - DNA; RNase - RNA; Some do both•Exonuclease - starts at an end•Endonuclease - cuts internally•Products may be oligonucleotides or nucleoside phosphates•Most are relatively nonspecificRestriction Endonucleases“Restriction Enzymes”•Recognize specific sequences in DNA•Many cut specifically at the recognition sequence•Products are relatively large polynucleotides•Some restriction enzymes produce products with “sticky ends”Restriction Endonucleases (= “Restriction Enzyme”)•Recognition sites vary in length•Short recognition sequences occur frequently in DNA, so enzymes with short recognition sequences produce a large number of small fragments•Long recognition sequences are rare, so enzymes with long recognition sequences produce a small number of large fragmentsRestriction Enzymes•Restriction enzyme recognition sites are often palindromes:–A palindrome or palindromic sequence is the same forward on one strand and backwards on the other.– 5’ GAATTC 3’ 3’ CTTAAG 5’•“Madam I’m Adam.”•There are 4 possible bases that can occur at any given position•Therefore the recognition site for a “6-cutter” like EcoRI is found 1 in 46 bp or 1 in 4096 bp ON AVERAGE1/4 nExamples of Restriction EnzymesEcoRI site-OH3’‘3HO-P5’’5P•Each of these enzymes recognizes a specific 6 bp palindromic sequence.•HpaI produces blunt ended fragments.•EcoRI, HindIII, and PstI produce fragments with sticky ends.Figs 7-4 7-5Why “Restriction” Endonuclease? •Bacteria possess enzymes that “restrict” incoming “foreign” DNA (like bacteriophage DNA) by cleaving it. •If a bacterial cell contains a “restriction endonuclease” to inactivate invading phage DNA, how does it avoid attacking its own DNA?•Answer: Modification: It modifies (by methylation) every recognition site in its own DNA.•The restriction endonuclease can’t recognize the modified (methylated) sites.•Nearly all bacterial genomes encode one or more “restriction/modification” systems.•Scientists and now companies have cloned the genes for these enzymes, overproduced the proteins, purified them, and sell them for lots of money. See www.neb.com for example.CH3Uses of Restriction Enzymes•Cloning–Any two pieces of DNA with compatible ends can be easily ligated together.–Modern techniques make it easy to add restriction sites to any piece of DNA.•Analysis–The relative position of restriction sites provides information about the sequence of the DNA.–Restriction analysis allows easy comparison of two different DNAs or confirmation of cloning events.DNA Separation by Gel ElectrophoresisPurposes•Analytical tool- To identify or learn something about a particular piece of DNA: size, topology, restriction pattern, purity, concentration •Preparative tool - To separate a fragment of interest from a complex mixture of DNA fragmentsSeparation of DNA Fragments by Gel ElectrophoresisFig 7-1Gel ElectrophoresisThe Gel Sieves the Molecules -Smaller Molecules Migrate Faster+-TimeHigh MW DNA, increased frictional draglow MW DNA, decreased frictional dragGel Electrophoresis•Charged molecules will move in an electric field.•A gel matrix is used to “sieve” the molecules–Agarose – loose; broad separation range –Acrylamide – tighter; narrow separation range•Movement through the gel is dependent on charge and shape.•In order for movement to be proportional to mass (molecular weight) then the molecule must have a constant charge to mass ratio.Gel Electrophoresis•DNA has 2 negative charges (phosphates) per base pair •All linear double stranded DNA molecules have (essentially) the same shape•Therefore – movement is proportional to length (MW)–Distance µ 1/logMW+A B C D EDNA loaded in wellsMovement of DNAfragmentsGel ElectrophoresisEthidium bromide can be used to visualize DNA fragments after electrophoresisEthidium bromide fluoresces. When it absorbs UV light it emits visible light. When it is bound to DNA its fluorescence intensity increases about 20 fold.Fluorescence excitation and emission spectra of ethidium bromide bound to DNAExcitation spectrum - the light that goes in from a