Essential Elements of Biochemistry BCHM 3050 Dr Srikripa Chandrasekaran Lecture 2 16 15 Notes Structure Function of DNA RNA Nucleic Acids Continued I Hershey and Chase Experiment A Viruses can infect humans animals plants bacteria etc B They used a virus and bacteria C Virus injects something inside of the host cell reconstitute into a new virus inside the cell D Cover of the virus is the protein green and red is the DNA E DNA has phosphorus nitrogen oxygen F Protein has all of these plus sulfur G Bacteria is heavier than the virus so the bacteria will collect at the bottom in a pellet H The phosphorus label should show up in the bacteria and it did I The radioactive sulfur remained on top and the radioactive phosphorus entered the pellet J Elements present in DNA phosphorus nitrogen carbon oxygen K Elements in proteins sulfur nitrogen carbon oxygen L Only difference was the presence of phosphorus versus sulfur M Tracking DNA in one vile and protein in another N Bacteria divides every 20 minutes O Spin everything in a blender bacteria is heavier so it settles to the P bottom supernate material lies on the top If DNA infected bacteria the phosphorus label should be in the pellet at the bottom of the vile Q Results of the experiment 1 Bacteria pellet had radioactive phosphorus 2 Protein s radioactive sulfur remained on the top in the supernate 3 Genes consist of DNA genetic break through Information Processing DNA RNA Protein Traits A The 4 bases are the same in eukaryotes and prokaryotes B Bases code for certain proteins C Even identical twins do not have the same exact DNA over time as it is altered by the environment D Process by which DNA is converted to RNA is transcription E RNA Protein occurs via translation Variation in DNA Structure A Palindrome should read the same top on the top and the bottom strand can be slightly staggered B Palindromes produce the cross like DNA structures EcoR1 Restriction Site II III IV 1 V VI A Recognize palindrome cut palindrome create gaps Sticky ends in DNA sequence of the host 1 DNA is now opened to allow bacteria to come in a integrate into the genome of the host DNA B Restriction enzymes cut the palindrome Bacterial Plasmid Vector for Cloning of Foreign DNA A Circular piece of DNA in bacteria plasmid B Plasmids have many palindromes where enzymes can come in a cut the DNA that allow them to change and adapt DNA necessary for the creation of drugs etc C Plasmids also have certain genes D Modified genes can be inserted in plasmids DNA Gel electrophoresis A DNA is not visible even under a microscope B Buffer conducts electricity C Make use of the negative charge of DNA D Negative charge at one end of the gel and positive charge at the other end E When electricity is turned on the DNA will migrate towards the positive end of the gel DNA is separated in agarose F Ethidium bromide carcinogen compound that can bind to DNA on the minor group stick to it and when shine UV light the ethidium bromide with pick up the light and shine white light G Agarose is used to make a gel to attract DNA in it H Lane 7 contains a marker used as a comparison to determine the molecular weights of the other DNA pieces used for comparison purposes I Smallest pieces of DNA will move the farther fastest J DNA will separate according to size largest will stay on top and smallest with move to the bottom VII Problem 1 A Enzyme 1 in lane 1 should recognize a certain set of palindromes Enzyme 2 recognizes different set of palindromes etc 1 Same plasmid is digested in each lane just with different restriction enzymes that cut at different restriction sites 2 Add up the sizes where the bands occur to get the overall size of a plasmid the two pieces in lane 1 and lane 2 should each add up to around 7 5kb 3 Obviously something went wrong in lane 3 4 Lane 4 if have 2 pieces that are the same size they will sit on top of each other in the gel causing the appearance of a fat band VIII Problem 2 A 1000 base pairs total B The areas where the enzymes are marked are where the palindromes occur and the enzyme will cut at these locations C BamH1 will make 4 cuts 2 1 Sizes of the 4 pieces 100bp 150bp 100bp 650 bp 150 500 they should all add up to 1000 2 EcoR1 will create 3 pieces after digestion Sizes 350bp 200bp 450bp 100 350 IX Sanger Sequencing A Often pieces on the top of the gel are thicker than those on the bottom but if you see a thick band on the bottom then it is likely that two pieces are overlapping each other B Palindrome is recognized by the restriction site 3
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