RENATURATION KINETICS - 1 fast renaturing repetitive...

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RENATURATION KINETICS Study of the rate at which denatured DNA (ssDNA) renatures. Visualize a long DNA molecule 100 base pairs in length 1...ATC. ..ATC. ...ATC. ..... ATC. ....ATC. ...100 1...TAG. ..TAG. ...TAG. ..... TAG. ...GAG. ..100 Interspersed sequence of 3 base pairs in length (ATC) repeated sequence represents 15% of the total DNA [3% X 5 = 15%] 1. Now, if you cleave the DNA into very small segments 2. denature the segments into single strands 3. allow the strands to renature over time 4. moniter renaturation by measuring the change in absorbance at 260nm A graph of the results is shown in Fig. 2 . Interpretation: 2 different classes of fragments
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Unformatted text preview: 1) fast renaturing repetitive fragments ~15% of the DNA 2) slow renaturing - unique sequences, non-repetitive ~85% of the DNA Repeated DNA sequences reanneal faster than the unique sequences of DNA because they are present at a higher concentration. Assumption in this type of analysis: the slowest fraction represents fragments of DNA for which there is only one copy. If you perform the experiment with DNA fragments in which there are no repeating sequences you get a single, slow reannealing curve as shown in Fig. 3 . All renaturation will occur at the same rate...
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