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Lecture 32

BIO 201 Lecture 32: Structure and Replication of DNA

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University at Buffalo
Biological Sciences
BIO 201
Lara Hutson

BIO 201 Dr. Lara Hutson Lecture 32 Notes 4/26/17 How is DNA Replicated? ● Semi-conservative ○ Parental strand is used as template for creation of new daughter strand ○ Each daughter cell inheriting half old, half new DNA ● Conservative ○ Double stranded parental DNA is retained in its entirety through replication and inherited by one of daughter cells ○ Cell reads double stranded sequence to create an entirely new copy of DNA that is in sequence identical ● Dispersive ○ DNA replicated in little bits and glued back together again ○ Comprised of old and new DNA The Meselson-Stahl Experiment (1958) ● Discovered mechanisms of replication ○ Labeled DNA during replication with different isotopes of nitrogen (different densities) ○ Purify DNA every generation ○ Identify density of DNA at each generation using density gradient centrifugation! ● Differential centrifugation ○ Particles whose mass is above a certain size pellet at selected centrifugal force ○ Separate based on mass (= size) ○ Good for separation of structures with very different masses ● Density gradient centrifugation ○ Suspension is a solute concentration gradient ○ Particles settle where particle density matches solute density ○ Separate based on density ○ Better for separation of similarly-sized things ○ Requires very high speeds/special equipment Density Gradient Centrifugation of DNA 1. Extract DNA 2. Place in tube with CsCl gradient 3. Centrifuge at 140,000g 4. Observe DNA with UV light a. DNA migrates to location where its density matches CsCl density b. Spin for ~48 hrs to get DNA to coalesce to a narrow band in tube that corresponds to [ ] CsCl Changing Nitrogen Isotopes Changes Density of DNA 15 1. Feed E. coli N (heavy N) for several generations 2. Purify DNA from N-fed E. coli and fro
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