Question

Not just for enzymes. Kinetics is useful for studying reactions of all types, not just those catalyzed by enzymes. In Chapters 4 and 5, we learned that DNA could be reversibly melted. When melted double-stranded DNA is allowed to renature, the process can be described as consisting of two steps, a slow second order reaction followed by a rapid first order reaction. Explain what is occurring in each step.

Answer 1

Ans. DNA Renaturation Kinetics

C₀t analysis, a technique which is based on the principles of DNA reassociation kinetics, is a biochemical technique that measures how much repetitive DNA are present in a genome. It is also used to study genome structure and organization.

When double-stranded DNA is denatured and allowed to renature, the rate of renaturation can give valuable information about the complexity of the DNA if there are repetitive sequences in the DNA, it shows less complexity in comparison to its total length, but the complexity is equal to its total length if all sequences are unique.

The rate of renaturation of fully denatured DNA is kinetically a second-order reaction. If the extent of renaturation is plotted against log C₀t (known as Cot curve), it is observed that part of the DNA is renatured quite rapidly while the rest is very slow to renature. This indicates that some sequences have a higher concentration than others i.e., part of the genome consists of repetitive sequences.

When melted double-stranded DNA is allowed to renature, the process can be described as consisting of two steps, a slow second order reaction followed by a rapid first order reaction.

If the reformation of helical structures arises from the interaction of two separate complementary chains, the kinetics of the reaction should be second order; on the other hand, they should be first order if the strands were never completely separate following the structural collapse. In the latter case, the polynucleotide chains of the denatured molecules may be imagined as only partially separated, being held together by entanglements and, at least at lower temperatures, some randomly formed hydrogen bonds. Renaturation would then represent a zippering-up of a single kinetic entity.

The given below figure shows the dependence of renaturation on the C₀ t values.