Essential Elements of Biochemistry BCHM 3050 Dr Srikripa Chandrasekaran Lecture 2 20 15 Notes Nucleic Acids Part 2 I 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 enzyme and the restriction enzyme cuts it and creates sticky ends at the palindrome C DNA double strands are complementary to each other D Phosphodiester bonds exist between nucleotides E Need an OH group at the 3 end F Sanger generated dideoxy versions of nucleotides ddGTP ddCTP ddTTP ddATP Sanger replaced the OH group with just an H so instead of having an OH group and an H group the nucleotides just have 2 Hs 1 He splits the DNA strands apart and adds one strand of DNA in a tube cannot choose both strands 2 Has 4 tubes 1 adds 1 strand of DNA dATP CTP GTP TTP ddATP 2 adds 1 strand of DNA dATP CTP GTP TTP ddGTP 3 adds 1 strand of DNA dATP CTP GTP TTP ddCTP 4 adds 1 strand of DNA dATP CTP GTP TTP ddTTP These di deoxynucleotides prevent the DNA chains from being able to extend Radioactively labeled the phosphate groups of the dd versions of the nucleotides He is trying to recreate the bottom strand of the original DNA Read from 5 to 3 on a gel read bottom to top bottom smallest top largest A dd dioxyribonucleotide cannot synthesize DNA The gel shows the complimentary strand because Sanger wanted to recreate the complimentary strand II G Can use color code to signify different nucleotides and sequence a DNA strand each color signifies a specific nitrogenous base Southern Blotting A The usage of this procedure has decreased in recent years because it is very cumbersome and there are more efficient ways to obtain the information that this process leads to B For a long time this was a very widely used procedure C A guy with the last name Southern came up with this procedure 1 D Uses restriction enzymes to cut up large strand of DNA into tiny pieces E Two pieces of DNA that he is interested in are darkly stained F Run all pieces of DNA in agrose gel 1 Figures our where DNA of interest is by sending a probe which is the actually sequence of DNA labels it and adds it to the DNA gel 2 Can also put filter paper on top of gel and filter paper will absorb all pieces of DNA through adhesion 3 Send radioactive probe and wherever the DNA of interest is the 4 Southern Blotting is not very helpful but can tell you if your DNA is probe will light up there or if it is not there 5 Main principle of this technique just determining whether or not your piece of DNA of interest is there or not 2
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