Denaturation and renaturation of DNA

Figure 24.26 gp32 facilitation of both denaturation and renaturation of DNA. 

Steps in denaturation and renaturation of a DNA duplex. In step l the temperature is raised to the point where the two strands of the duplex separate. If denatured DNA is slowly cooled, the events depicted as steps 2 and 3 follow. In step 2 a second-order reaction occurs in which two complementary strands of DNA must collide and form interstrand hydrogen bonds over a limited region. Step 3 is a first-order reaction in which additional hydrogen bonds form between the complementary strands that are partially hydrogen-bonded (zippering). Once complementary strands are partially bonded, the zippering reaction occurs rapidly. In the overall process, step 2 is rate-limiting. 

Denaturation and Renaturation When DNA molecules are heated to certain temperature (e.g., lOO C), the two polynucleotide strands separate. The transition from the double strand (original form) to the single strand (denatured form) can be observed by the change in optical density at 260 nm. The plot of the optical density versus temperature gives a sinusoidal curve that is similar to an acid-base titration curve. In Figure 17.8 the point Tin, which corresponds to the equivalence point in an acid-base titration, is the hypochromic point and denotes where a mixture of the native and denatured strands occurs. 

The circular genetic map of the T-even phages would be very easy to understand if it could be assumed that the DNA molecules of these phages are circular in structure. However, native DNA molecules of phage T2 are linear in structure on electron micrographs. After denaturation and renaturation, the DNA molecules... 

Kinetic analysis indicates that renaturation is a two-step process. In the slow step effective contact is made between two complementary regions of DNA originated from separate strands. This rate-limiting step called nucleation is a function of the concentration of complementary strands. Nucleation is followed by a relatively rapid zippering up of adjoining base residues into a duplex structure. The steps involved in denaturation and renaturation are depicted in figure 25.14. 

Eigner, J.-. Native, denatured and renatured states of DNA. Doctoral Thesis, Department of Chemistry. Harvard University, Cambridge, Mass., April 1960. 

The double helix is a relatively stiff and elongated molecule. Consequently, a solution of DNA has a high viscosity. If such a solution is heated to 95°C, the viscosity drops markedly, reflecting a collapse of the double-helical structure. This is known as denaturation and is accompanied by separation of the duplex into its single strands, which are fairly flexible. Denaturation and renaturation provide valuable information on important properties of the DNA obtained from various sources. Denaturation also provides the basis for very precise and sensitive approaches to the identification of specific sequences in both DNA and RNA. This has been central to the rapid developments in molecular genetics. 

The study of denaturation and renaturation patterns of DNA is an useful tool to analyze the stabilizing and destabilizing actions of metal ions (6), the formation of cross links (7) or single strand breaks (8). 

Such renaturation or annealing of complementary DNA strands is an important step in probing a Southern blot and in performing the polymerase chain reaction (reviewed in Chapter 7). In these techniques, a well-characterized probe DNA is added to a mixture of target DNA molecules. The mixed sample is denatured and then renatured, When probe DNA binds to target DNA sequences of sufScient complementarity, the process is called hybridization. 

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