Lecture 121 nucleotide (DNA)=- 1 nitrogenous base (adenine, thymine, cytosine, or guanine)- 1 5-carbon sugar (deoxyribose)- 1 or more phosphate group(s)Base is attached to 1’ carbonPyrimidines: Cytosine, Thymine, UracilPurines: Adenine, Guanine5’ to 3’5’ = the number 5 carbon of the (deoxy)ribose -bears the phosphate group3’ = the number 3 carbon of the (deoxy)ribose -bears a hydroxyl (-OH) groupDNA strands are antiparallelBases pair by hydrogen bondingA-T: 2 bondsG-C: 3 bondsIt takes more energy to break G-CChargaff’s Rule• DNA from any cell of all organisms should have a 1:1 ratio of pyrimidine and purine bases andthe amount of guanine is equal to cytosine and the amount of adenine is equal to thymine.• Given the proportion of just one base, the proportions of all other bases can be calculated.DNA replication is semiconservative and bidirectionalDNA polymerase can only synthesize nucleotides in the 5’ to 3’ directionDNA polymerase requires a primer1.Helicase breaks hydrogen bonds. Topoisomerase relaxes supercoiling2.Single-stranded binding (SSB) protein prevents reannealing3.DnaG synthesizes RNA primers4.DNA polymerase III synthesizes daughter strand5.DNA polymerase III elongates the leading strand continuously and the lagging stranddiscontinuously6.DNA polymerase I removes and replaces nucleotides of the RNA primer7.DNA ligase joins Okazaki fragmentsLeading strand polymerase moves in the same direction as the replication fork; lagging strandpolymerases move in the opposite direction.
DNA polymerase III holoenzyme holds leading and lagging strand synthesis together at thereplication fork.ENZYMES●DNA Topoisomerase- relaxes supercoiling●Helicase (DnaB)- unwinds the double helix●SSB- prevents reannealing of separated strands●Primase- synthesizes RNA primers●DNA pol III- synthesizes DNA●DNA pol I- removes and replaces RNA primer with DNA●DNA ligase- joins DNA segmentsLecture 13polymerase chain reaction• “replication in a tube"• replicate short fragments of DNA (= amplification)• use repeated sequence of temperatures (= cycle)reaction ingredients1. DNA template with region to be copied (amplified)2. deoxynucleotide triphosphates = dNTPs (dCTP, dTTP, dATP, dGTP)3. DNA polymerase (usually Taq)4. aqueous buffer (including MgCl2)5. DNA primerswhat is a primer?• short piece of DNA of known sequence• complementary to target DNA sequence1.Denaturation (95C)2.Annealing (~50C)3.Extension (72C)PCR amplification• the number of molecules doubles each cycle• the number of molecules after n cycles is 2n• e.g. 22 = 2x2 = 4Sanger sequencingsequencing reaction ingredients1. DNA template (typically PCR product)2. deoxynucleotide triphosphates = dNTPs (dCTP, dTTP, dATP, dGTP)
3. aqueous buffer (including MgCl2)4. polymerase (usually Taq)5. DNA primers
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