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Lecture 6

BIO372 - Lecture 6.pdf

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University of Toronto Mississauga
Gordon Anderson

Lecture 6 January-28-14 10:12 AM Hypotheses of DNA replication Red is new DNA, blue is old DNA Separation of DNAs of different density by CsCl density gradient centrifugation Density gradient centrifugation Analytical approach - they note position of DNA in the float Two peaks of DNA We need to label the DNA Hypotheses of DNA replication Meselson and Stahl experiment to distinguish among the three hypothesis. They labeled E. coli DNA with N14 and 15 - if we have heavy N, DNA will be heavier Lighter isotope acts as precursor We watch DNA to be lighter over time CsCl gradient ultracentrifugation determines the density of the DNA. Heavy and light DNA will separate Completelyunlabeled = red (light DNA) in 2nd line - will be moreintense, blue will be less intense Conservative - We end up with light DNA, no heavy DNA left Dispersive - the II mixed will be positioned in between light and heavy DNA Density scans from Meselson and Stahl 0.3 generations? The cells are in synthesis phase, someare later some are earlier How are there intermediates?This is an average value, About 20 min to divide cell which is too big Dispersive goes down (which means it doesn’t happen) The lightest possible band always darken in the next generation We have a reference point so we can comparethe generations to each other It doesn’t matter how much DNA is loaded in each gen, the pattern is very clear so we can conclude that it is semi-conservative Three possible models of DNA replication: which is correct? Continuous - having two different enzymes for each strand replication fork is the start of replication Semidiscontinuous - a series of fragments are created Discontinuous - two strands create fragments Evidence for short DNA fragments in replicatingT4 DNA - Okazaki fragments One of the precursors are radiolabeled We look for pattern that incorporates the radioactivity Where is the radioactivity in the tube? We have centrifuge tube and poke a hole, each tube will drip, we'll see the radioactivityhere 30, 60, 120 secs. The peak goes higher - tells us that we have long fragments - synthesis is discontinuous Instead of T, we have U in DNA - we'll have repair sites - break DNA at that spot and therefore, there will be short fragments We need to a mutant that is defective in some process - we need to knock the repair so if we don’t break DNA with U Evidence for short fragments in replicatingT4 DNA in a ligase deficient mutant - Okazaki fragments persist Primingin DNA synthesis DNA need to have a primer to start synthesis - we have RNA primers, how to detect them? RNA can get started without a primer Identification o
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