What is the Difference Between Denaturation and Renaturation of DNA

The main difference between denaturation and renaturation of DNA is that denaturation of DNA is the process of separating dsDNA into single strands. But, in contrast, renaturation of DNA is the process of forming base pairs; that is, coming back together of the complementary DNA strands. Furthermore, hydrogen bonds are broken down during denaturation of DNA, but hydrogen bonds form between complementary bases during the renaturation of DNA. Moreover, the main factors affecting the denaturation of DNA are the physical agents, such as heating and sonication, and chemical agents, such as alkaline, formamide, and DMSO. Meanwhile, the factors, affecting renaturation are the ionic strength of the solution, temperature, time, DNA concentration, and the size of the interacting molecule. 

In brief, denaturation and renaturation of DNA are two processes of hydrogen bond breaking and remaking in DNA. Generally, they are important in the number of bioassays, which involve DNA hybridization, such as membrane hybridization, microarrays, PCR, etc. 

Key Areas Covered 

1. What is the Denaturation of DNA
     – Definition, Process, Importance
2. What is the Renaturation of DNA
     – Definition, Process, Importance
3. What are the Similarities Between Denaturation and Renaturation of DNA
     – Outline of Common Features
4. What is the Difference Between Denaturation and Renaturation of DNA
     – Comparison of Key Differences

Key Terms 

Absorbance, Chemical Denaturation, Denaturation of DNA, dsDNA, Physical Denaturation, Renaturation of DNA, ssDNA, Temperature

What is the Denaturation of DNA 

Denaturation of DNA is the process of separating double-stranded DNA into two single strands. It occurs as a result of breaking hydrogen bonds. Besides, the denaturation of the double helix can occur in two methods; the physical denaturation and chemical denaturation.  

Physical Denaturation  

Physical denaturation is mainly done by heating and sonication. Usually, high temperatures above 90°C result in the denaturation of DNA. Technically, this is done by providing enough kinetic energy to breakdown hydrogen bonds between the base pairs. Subsequently, this results in the disruption of Watson and Crick base pairs and the separation of the double-stranded helix into two single strands. In this process, the characteristic temperature that melts the DNA double helix is known as the melting temperature (Tm). At this temperature, 50% of DNA is melted. Also, Tm increases with the increasing G-C content as guanine and cytosine form three hydrogen bonds during base pairing, while cytosine and thymine form only two hydrogen bonds. Generally, the degree of denaturation can be spectrophotometrically determined by monitoring the absorbance of light at 260 nm.

Sonication is another method of denaturation. In this, probe sonication and sonication bath are two techniques of sonication.

Figure 1: Denaturation and Renaturation in PCR

Chemical Denaturation 

Concerning chemical denaturation, the presence of chemical agents, such as urea and formamide, accelerates denaturation by stabilizing purines and pyrimidines. Thus, they decrease the Tm. For example, 95% of formamide completely denatures DNA at room temperature. Additionally, various concentrations of sodium hydroxide and DMSO also decreases the melting temperature of DNA. 

What is the Renaturation of DNA 

Renaturation of DNA is the process of annealing the two complementary DNA strands together. Eventually, it occurs with cooling. Technically, renaturation happens through the reformation of hydrogen bonds between complementary base pairs, which in turn brings the two DNA strands together to form the double-stranded DNA. Furthermore, the renaturation of DNA can be monitored by monitoring the absorbance. In general, the absorbance of DNA at 260 nm decreases while renaturing the DNA. Besides, the degree of renaturation depends on C0, which is the concentration of double-stranded DNA prior to denaturation, and t, which is the time taken to renaturation.  

Moreover, the renaturation of DNA is quite rapid up to some point while the rest will be slow. Significantly, this indicates that some sequences have a higher concentration in the genome; for example, repetitive sequences. Therefore, the rate of renaturation gives important information about the complexity of the genome. 

Similarities Between Denaturation and Renaturation of DNA  

• Denaturation and renaturation of DNA are two processes the complementary DNA strands undergo.  
• Generally,  DNA is a double-stranded molecule, containing two strands with complementary base pairs, which run in an antiparallel manner.  
• These complementary base pairs form hydrogen bonds with each other to hold the two DNA strands together.  
• During the denaturation and renaturation processes, breakdown and formation of hydrogen bonds can be identified depending on the chemical and physical conditions of the solution.  
• Both processes are important in DNA hybridization assays, such as DNA  microarrays, membrane hybridization, etc. as well as, in PCR. 

Difference Between Denaturation and Renaturation of DNA 

Definition 

Denaturation of DNA refers to the unwinding of the double-stranded DNA by the breaking down of hydrogen bonds, which hold the two DNA strands together. In contrast, renaturation of DNA refers to the formation of base pairs; that is, it refers to the two complementary strands of the DNA coming back together. 

Give Rise to 

Denaturation of DNA gives rise to single-stranded DNA, but the renaturation of DNA gives rise to double-stranded DNA. 

Hydrogen Bonds 

Denaturation breaks down hydrogen bonds between complementary base pairs, but in contrast, renaturation forms hydrogen bonds between complementary base pairs. 

Factors Affecting the Processes

The main factors affecting the denaturation of DNA are the physical agents, such as heating and sonication, and chemical agents, such as alkaline, formamide, and DMSO. On the other hand, the factors affecting renaturation are the ionic strength of the solution, temperature, time, DNA concentration, and the size of the interacting molecule. 

Effect on the Absorbance 

Denaturation increases the absorbance of DNA at 260 nm, but renaturation decreases the absorbance of DNA at 260 nm. 

Effect on the Optical Rotation 

Denaturation highly decreases the positive optical rotation of the DNA double helix, while renaturation gives a strong, positive optical rotation to the DNA double helix. 

Effect on the Viscosity 

Denaturation remarkably decreases the viscosity of DNA, but renaturation highly increases the viscosity of DNA. 

Conclusion 

Denaturation of DNA is the process of breaking down of hydrogen bonds of the DNA, and thus, separating the DNA duplex into two single-stranded DNA. Significantly, it increases the absorbance of DNA at 260 nm while decreasing the positive optical rotation and the viscosity. In contrast, the renaturation of DNA is the process of remaking the hydrogen bonds between the two complementary DNA strands, in order to form the double-stranded DNA. Generally, it decreases the absorbance at 260 nm, while increasing the viscosity and the positive optical rotation. Therefore, the main difference between denaturation and renaturation of DNA is the behavior of hydrogen bonds in the DNA and the effects. 

References:

1. Shah, Richa. “Denaturation and Renaturation of DNA.” Biology Discussion, 2 May 2016, Available Here.
2. Wang, Xiaofang et al. “Characterization of denaturation and renaturation of DNA for DNA hybridization.” Environmental health and toxicology vol. 29 e2014007. 11 Sep. 2014, doi:10.5620/eht.2014.29.e2014007.

Image Courtesy:

1. “PCR Steps” By Tinojasontran – Own work (Public Domain) via Commons Wikimedia