Cot Analysis
Cot Analysis of DNA Renaturation (single transition) In a DNA renaturation experiment the concentration of single strands remaining as a function of time is found to be 2nd order in single-strand concentration C. Integration between t = 0 and t yields: or which can be expressed as: Here C/Co is the fraction of the single strands remaining. Note that when kCot = 1, t > t1/2 = 1/kCo and k = 1/Cot1/2 At that point, C/Co = 0.
5, or one half of the DNA is in the single-stranded form.The equation above could be expressed as: In terms of the fraction of the DNA renatured, fren, we have: Eq. 6 A DNA is allowed to renature for a time t, and the fraction that has renatured (fren) during that time is measured. Knowing the initial [DNA], Co, the product Cot, referred to as the “Cot” can now be used to obtain k. From above, k can then be related to the “Cot1/2”: Cot1/2 = 1/ k The “Cot1/2” which is a constant (1/k) for a particular DNA could be obtained directly from the midpoint of the renturation curve, i.
e. f you find the time precisely for 50% renaturaton. It can be calculated, however, from the time required for any value of fren along the curve. Equation 6. can be expressed as: From the data below for T4 DNA, fren = 0.
436 (43. 6% renatured) at Cot = 0. 325 Ms. This Cot value was obtained from the product of t = 65 s and a Co = 5×10-3 M. You should be able to calculate a Cot1/2 value for T4 which compared with that of E coli (9. 4 Ms): , you should obtain the size of the T4 genome shown in the figure above.
You need to understand this analysis before moving on to that of a DNA (eukaryotic) of higher complexity which displays more than one renaturation transition. The difference in the Cot1/2 for T4 and E coli has nothing to do with the size of the DNA fragments that are undergoing renaturation. The DNA undergoing renaturation is of the same size. The DNA is sheared in a homogenizer to fragments that are about 450 bp in size. E coli DNA of the same physical size and concentration as that of T4 renatures more slowly since the concentration of complimentary partners is much lower.
